U.S. patent application number 12/091020 was filed with the patent office on 2008-10-30 for anti-il13 human antibodies.
This patent application is currently assigned to Novartis AG. Invention is credited to Joe Buechler, Emma Campbell, Sofia Parveen, Gunars Valkirs.
Application Number | 20080267959 12/091020 |
Document ID | / |
Family ID | 37908192 |
Filed Date | 2008-10-30 |
United States Patent
Application |
20080267959 |
Kind Code |
A1 |
Campbell; Emma ; et
al. |
October 30, 2008 |
Anti-Il13 Human Antibodies
Abstract
The present invention relates to human anti-IL-13 binding
molecules, particularly antibodies, and to methods for using
anti-IL-13 antibody molecules in diagnosis or treatment of IL-13
related disorders, such as asthma, atopic dermatitis, allergic
rhinitis, fibrosis, inflammatory bowel disease and Hodgkin's
lymphoma.
Inventors: |
Campbell; Emma; (West
Sussex, GB) ; Parveen; Sofia; (West Sussex, GB)
; Buechler; Joe; (Carlsbad, CA) ; Valkirs;
Gunars; (Escondido, CA) |
Correspondence
Address: |
NOVARTIS INSTITUTES FOR BIOMEDICAL RESEARCH, INC.
400 TECHNOLOGY SQUARE
CAMBRIDGE
MA
02139
US
|
Assignee: |
Novartis AG
|
Family ID: |
37908192 |
Appl. No.: |
12/091020 |
Filed: |
October 19, 2006 |
PCT Filed: |
October 19, 2006 |
PCT NO: |
PCT/EP2006/010098 |
371 Date: |
April 22, 2008 |
Current U.S.
Class: |
424/133.1 ;
424/158.1; 530/387.3; 530/389.2 |
Current CPC
Class: |
C07K 16/244 20130101;
A61P 37/08 20180101; A61P 29/00 20180101; C07K 2317/92 20130101;
A61K 38/012 20130101; A61P 35/02 20180101; A61P 35/00 20180101;
A61K 2039/507 20130101; A61P 11/06 20180101; A61P 17/00 20180101;
A61P 1/04 20180101; A61P 37/00 20180101; C07K 2317/21 20130101;
A61P 11/02 20180101; Y10S 514/853 20130101; C12P 21/02 20130101;
A61K 2039/505 20130101; C12N 15/81 20130101; C07K 2317/76
20130101 |
Class at
Publication: |
424/133.1 ;
530/389.2; 530/387.3; 424/158.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/24 20060101 C07K016/24; A61P 11/06 20060101
A61P011/06 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 21, 2005 |
GB |
0521509.0 |
Aug 22, 2006 |
GB |
0616666.4 |
Claims
1-26. (canceled)
27. An isolated antigen-binding region of an antibody or functional
fragment thereof, comprising an H-CDR3 region depicted in an amino
acid sequence selected from SEQ ID NO: 9 and SEQ ID NO: 10, and
conservative variants thereof.
28. The isolated antigen-binding region according to claim 27,
where the H-CDR1, H-CDR2 and H-CDR3 and L-CDR1, L-CDR2 and L-CDR3
regions depicted in an amino acid sequence together are selected
form (i)-(iii): (i) SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, and
SEQ ID NO: 16, SEQ ID NO: 19, SEQ ID NO: 20; (ii) SEQ ID NO: 7, SEQ
ID NO: 8, SEQ ID NO: 10, and SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID
NO: 21; and (iii) SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, and
SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 22; and conservative
variants thereof.
29. An isolated antigen-binding region comprising a sequence having
at least 60, 70, 80, 90, or 95 percent sequence identity in the CDR
regions with the CDR regions described in claim 27.
30. An isolated human or humanized IL 13 antibody comprising an
isolated antigen-binding region according to claim 17.
31. An antibody according to claim 30 which comprises at least one
antigen binding site comprising an HC domain having an amino acid
sequence selected from any one of SEQ ID NO: 23, 27, 31 and 35, or
a sequence having at least 60, 70, 80, 90 or 95 percent sequence
identity thereof.
32. An antibody according to claim 30 which comprises at least one
antigen binding site comprising an LC domain having the amino acid
sequence selected from any one of SEQ ID NO: 25, 29, 33 and 37, or
a sequence having at least 60, 70, 80, or 95 percent sequence
identity thereof.
33. An antibody that comprises at least one antigen binding site
comprising a first HC domain having an amino acid sequence selected
from any one of SEQ ID NO: 23, 27, 31 and 35, or a sequence having
at least 60, 70, 80, 90 or 95 percent sequence identity thereof,
and a second LC domain having the amino acid sequence selected from
any one of SEQ ID NO: 25, 29, 33 and 37, or a sequence having at
least 60, 70, 80, 90 or 95 percent sequence identity thereof.
34. An antibody according to claim 30 comprising an HC variable
region according to SEQ ID NO: 23 and an LC variable region
according to SEQ ID NO:25, or a sequence having at least 60, 70,
80, 90 or 95 percent sequence identity thereof.
35. An antibody according to claim 30 comprising an HC variable
region according to SEQ ID NO: 27 and an LC variable region
according to SEQ ID NO: 29, or a sequence having at least 60, 70,
80, 90 or 95 percent sequence identity thereof.
36. An antibody according to claim 30 comprising an HC variable
region according to SEQ ID NO: 31 and an LC variable region
according to SEQ ID NO: 33, or a sequence having at least 60, 70,
80, 90 or 95 percent sequence identity thereof.
37. An antibody according to claim 30 comprising an HC variable
region according to SEQ ID NO: 35 and an LC variable region
according to SEQ ID NO: 37, or a sequence having at least 60, 70,
80, 90 or 95 percent sequence identity thereof.
38. An antibody according to claim 30, which is an IgG1 or an
IgG4.
39. A pharmaceutical composition comprising an antibody or
functional fragment thereof according to claim 30 and a
pharmaceutically acceptable carrier or excipient therefor.
40. A method for treating a disorder or condition associated with
the presence of cell receptor target IL-13, comprising
administering to a subject in need thereof an effective amount of
the pharmaceutical composition according to claim 39.
41. The method according to claim 40, wherein the disorder or
condition is asthma.
Description
FIELD OF USE
[0001] The present invention relates to specific binding members,
in particular human anti-IL-13 antibody molecules and especially
those which neutralize IL-13 activity. It further relates to
methods for using anti-IL-13 antibody molecules in diagnosis or
treatment of IL-13 related disorders, such as asthma, atopic
dermatitis, allergic rhinitis, fibrosis, inflammatory bowel disease
and Hodgkin's lymphoma.
BACKGROUND OF THE INVENTION
[0002] Interleukin (IL)-13 is a 114 amino acid cytokine with an
unmodified molecular mass of approximately 12 kDa [McKenzie, A. N.,
et al. J Immunol, 1993. 150 (12): p. 5436-44, and Minty, A., et al.
Nature, 1993. 362 (6417): p. 248-50.]. IL-13 is most closely
related to IL-4 with which it shares 30% sequence similarity at the
amino acid level. The human IL-13 gene is located on chromosome
5q31 adjacent to the IL-4 gene. This region of chromosome 5q
contains gene sequences for other Th2 lymphocyte derived cytokines
including GM-CSF and IL-5, whose levels together with IL-4 have
been shown to correlate with disease severity in asthmatics and
rodent models of allergic inflammation [Nakamura, Y., et al. Am J
Respir Cell Mol Biol, 1996. 15 (5): p. 680-7, Robinson, D. S., et
al. N Engl J Med, 1992. 326 (5): p. 298-304, Walker, C., et al. Am
J Respir Crit Care Med, 1994. 150 (4): p. 1038-48, Humbert, M., et
al. Am J Respir Crit Care Med, 1996, 154 (5): p. 1497-504,
Corrigan, C. J. and A. B. Kay Int Arch Allergy Appl Immunol, 1991.
94 (14): p. 270-1, Bentley, A. M., et al. Am J Respir Cell Mol
Biol, 1993.].
[0003] Although initially identified as a Th2 CD4+ lymphocyte
derived cytokine, IL-13 is also produced by Th1 CD4+ T-cells, CD8+
T lymphocytes NK cells, and non-T-cell populations such as mast
cells, basophils, eosinophils, macrophages, monocytes and airway
smooth muscle cells.
[0004] IL-13 is reported to mediate its effects through a receptor
system that includes the IL-4 receptor a chain (IL-4R.alpha.)-,
which itself can bind IL-4 but not IL-13, and at least two other
cell surface proteins, IL-13R.alpha..sub.1 and IL-13R.alpha..sub.2
[Murata, T., et al. Int J Hematol, 1999. 69(1): p. 13-20, Andrews,
A. L., et al. J Biol Chem, 2002. 277(48): p. 46073-8.].
IL-13R.alpha..sub.1 can bind IL-13 with low affinity, subsequently
recruiting IL-4R.alpha. to form a high affinity functional receptor
that signals [Miloux, B., et al. FEBS Lett, 1997. 401 (2-3): p.
163-6, Hilton, D. J., et al. Proc Natl Acad Sci USA, 1996. 93 (1):
p. 497-501]. The Genbank database lists the amino acid sequence and
the nucleic acid sequence of IL-13R.alpha..sub.1 as NP 001551 and
Y10659 respectively. Studies in STAT6 (signal transducer and
activator of transcription 6)-deficient mice have revealed that
IL-13, in a manner similar to IL-4, signals by utilizing the
JAK-STAT6 pathway [Kuperman, D., et al. J Exp Med, 1998. 187 (6):
p. 939-48, Nelms, K., et al. Annu Rev Immunol, 1999. 17: p.
701-38.]. IL-13R.alpha..sub.2 shares 37% sequence identity with
IL-13R.alpha..sub.1 at the amino acid level and binds IL-13 with
high affinity [Zhang, J. G., et al. J Biol Chem, 1997. 272 (14): p.
9474-80, Caput, D., et al. J Biol Chem, 1996. 271 (28): p.
16921-6.]. However, IL-13R.alpha..sub.2 has a shorter cytoplasmic
tail that lacks known signaling motifs. Cells expressing
IL-13R.alpha..sub.2 are not responsive to IL-13 even in the
presence of IL-4R.alpha. [Kawakami, K., et al. Blood, 2001. 97 (9):
p. 2673-9]. It is postulated, therefore, that IL-13R.alpha..sub.2
acts as a decoy receptor regulating IL-13 but not IL-4 function.
This is supported by studies in IL-13R.alpha..sub.2 deficient mice
whose phenotype was consistent with increased responsiveness to
IL-13 [Wood, N., et al. J Exp Med, 2003. 197 (6): p. 703-709,
Chiaramonte, M. G., et al. J Exp Med, 2003. 197 (6): p. 687-701].
The Genbank database lists the amino acid sequence and the nucleic
acid sequence of IL-13R.alpha..sub.2 as NP000631 and Y08768
respectively.
SUMMARY OF THE INVENTION
[0005] An embodiment of the invention herein provides an isolated
human or humanized antibody or functional fragment thereof with an
antigen-binding region that is specific for target protein IL-13
and the antibody or functional fragment thereof binds to IL-13. In
a related embodiment, the binding to IL-13 is determined at least
by cell surface IL-13 receptor binding preventing inflammatory
mediator release.
[0006] In still another embodiment, the invention provides an
isolated antigen-binding region of an antibody or functional
fragment thereof. In certain embodiments, the isolated
antigen-binding region includes an H-CDR3 region having an amino
acid sequence selected from SEQ ID NOs: 9-10, and conservative
variants thereof. As described herein, the conservative variants
include amino acid residues in any of the amino acid sequences
identified. In a related embodiment, the isolated antigen-binding
region is an H-CDR2 region having the amino acid sequence of SEQ ID
NO: 8, and conservative variants thereof. In another related
embodiment, the isolated antigen-binding region is an H-CDR1 region
having an amino acid sequence selected from SEQ ID NO: 6-7, and
conservative variants thereof.
[0007] In another embodiment, the isolated antigen-binding region
is an L-CDR3 region having an amino acid sequence selected from SEQ
ID NOs: 20-22, and conservative variants thereof. In still another
related embodiment, the isolated antigen-binding region is an
L-CDR1 region having an amino acid sequence selected from SEQ ID
NOs: 16-18, and conservative variants thereof. In yet another
related embodiment, the isolated antigen-binding region is an
L-CDR2 region having the amino acid sequence of SEQ ID NO: 19, and
conservative variants thereof.
[0008] In certain embodiments, the isolated antigen-binding region
is a variable light chain having an amino acid sequence selected
from SEQ ID 16-22, and conservative variants thereof.
[0009] In another embodiment, the isolated antigen-binding region
is a heavy chain having an amino acid sequence selected from one to
three of SEQ ID 6-10, and a sequence having at least 60, 70, 80, 90
or 95 percent sequence identity in the CDR regions with the CDR
regions having SEQ ID NOs: 6-10. In a related embodiment, the
isolated antigen-binding region is a light chain having an amino
acid sequence selected from one to three of SEQ ID NOs: 16-22, and
a sequence having at least 60, 70, 80, 90 or 95 percent sequence
identity in the CDR regions with the CDR regions having SEQ ID NOs:
16-22.
[0010] In a certain embodiment, the isolated antibody is an IgG. In
another embodiment, the isolated antibody is an IgG1 or an
IgG4.
[0011] In yet another embodiment, the invention provides an
isolated human or humanized antibody or functional fragment
thereof, having an antigen-binding region that is specific for an
epitope of IL-13, and the antibody or functional fragment binds to
IL-13 surface receptors on a cell. In a related embodiment, the
invention provides an isolated human or humanized antibody or
functional fragment thereof, having an antigen-binding region that
is specific for an epitope of target IL-13, and the epitope
contains one or more amino acid residues of amino acid residues
1-112 of target IL-13. In a related embodiment, the epitope is a
conformational epitope.
[0012] In yet another embodiment, the antibody or functional
fragment is a Fab or scFv antibody fragment. In a related
embodiment, the isolated antibody is an IgG. In another related
embodiment, the isolated antibody is an IgG1 or an IgG4.
[0013] In another embodiment, the invention provides a
pharmaceutical composition having at least one of any of the above
antibodies or functional fragments or conservative variants, and a
pharmaceutically acceptable carrier or excipient therefor.
[0014] In still another embodiment, the invention provides for a
transgenic animal carrying a gene encoding any of the above
antibodies or functional fragments thereof.
[0015] In certain embodiments, the invention provides a method for
treating a disorder or condition associated with the presence of a
cell having a receptor target for IL-13. The method involves
administering to a subject in need thereof an effective amount of
any of the above pharmaceutical compositions. In a related
embodiment, the disorder or condition to be treated is a
respiratory disorder.
[0016] In another embodiment, the disorder or condition to be
treated is bronchial asthma, which is a common persistent
inflammatory disease of the lung characterised by airways
hyper-responsiveness (AHR), mucus overproduction, fibrosis and
raised serum IgE levels. Li et al, Abstract for poster submitted at
The American Thoracics Society Annual Meeting, 2003, Seattle,
reported affects of a neutralising anti-mouse IL-13 antibody in a
chronic mouse model of asthma.
[0017] In another embodiment, the disorder or condition to be
treated is Chronic Obstructive Pulmonary Disease (COPD). Zheng et
al J Clin Invest, 2000. 106 (9): p. 1081-93, have demonstrated that
over expression of IL-13 in the mouse lung caused emphysema,
elevated mucus production and inflammation, reflecting aspects of
human COPD. mRNA levels of IL-13 have been shown to be higher in
autopsy tissue samples from subjects with a history of COPD when
compared to lung samples from subjects with no reported lung
disease (J. Elias, Oral communication at American Thoracic Society
Annual Meeting 2002). In another study, raised levels of IL-13 were
demonstrated by immunohistochemistry in peripheral lung sections
from COPD patients [Wardlaw, A. J., Clin Med, 2001. 1 (3): p.
214-8.].
[0018] In another embodiment, the disorder or condition to be
treated is selected from other inflammatory or obstructive airways
diseases and conditions such as acute lung injury (ALI),
acute/adult respiratory distress syndrome (ARDS), dyspnea, allergic
airway inflammation, small airway disease, lung carcinoma, acute
chest syndrome in patients with sickle cell disease and pulmonary
hypertension, as well as exacerbation of airways hyperreactivity
consequent to other drug therapy, in particular other inhaled drug
therapy.
[0019] In another embodiment, the disorder or condition to be
treated is bronchitis of whatever type or genesis including, e.g.,
acute, arachidic, catarrhal, croupus, chronic or phthinoid
bronchitis.
[0020] In another embodiment, the disorder or condition to be
treated includes pneumoconiosis (an inflammatory, commonly
occupational, disease of the lungs, frequently accompanied by
airways obstruction, whether chronic or acute, and occasioned by
repeated inhalation of dusts) of whatever type or genesis,
including, for example, aluminosis, anthracosis, asbestosis,
chalicosis, ptilosis, siderosis, silicosis, tabacosis and
byssinosis.
[0021] In another embodiment, the disorder or condition to be
treated is selected from atopic rhinitis (hay fever), allergic
dermatitis (eczema) and chronic sinusitis. Raised levels of IL-13
have been measured in human subjects with atopic rhinitis (hay
fever), allergic dermatitis (eczema) and chronic sinusitis. For
example levels of IL-13 were found to be higher in bronchial
biopsies, sputum and broncho-alveolar lavage (BAL) cells from
asthmatics compared to control subjects [Humbert, M., et al. J
Allergy Clin Immunol, 1997. 99 (5): p. 657-65, Kotsimbos, T. C., P.
Ernst, and Q. A. Hamid, Proc Assoc Am Physicians, 1996. 108 (5): p.
368-73, Komai-Koma, M., F. Y. Liew, and P. C. Wilkinson, J Immunol,
1995. 155 (3): p. 1110-6, Naseer, T., et al. Am J Respir Crit Care
Med, 1997].
[0022] In another embodiment, the disorder or condition to be
treated is selected from other inflammatory conditions of the skin,
for example, psoriasis or lupus erythematosus.
[0023] In another embodiment, the disorder or condition to be
treated is inflammatory bowel disease, such as ulcerative colitis
and Crohn's disease. Heller et al. (2002) Immunity, 17 (5): 629-38,
report that neutralisation of IL-13 by administration of soluble
IL-13Ra2 ameliorated colonic inflammation in a murine model of
human ulcerative colitis. Correspondingly, IL-13 expression was
higher in rectal biopsy specimens from ulcerative colitis patients
when compared to controls.
[0024] In another embodiment, the disorder or condition to be
treated is selected from other fibrotic conditions, such as
systemic sclerosis, pulmonary fibrosis, idiopathic pulmonary
fibrosis or fibroid lung. Increased levels of IL-13 have been
measured in the serum of patients with systemic sclerosis
[Hasegawa, M., et al. J Rheumatol, 1997. 24 (2): p. 328-32] and in
BAL samples from patients affected with other forms of pulmonary
fibrosis [Hancock, A., et al. Am J Respir Cell Mol Biol, 1998].
[0025] In another embodiment, the disorder or condition to be
treated is liver fibrosis. Specific inhibition of IL-13 by
administration of soluble IL-13Ra2 or IL-13 gene disruption, but
not ablation of IL-4 production prevented fibrogenesis in the liver
[Fallon, P. G., et al. J Immunol, 2000. 164 (5): p. 2585-91,
Chiaramonte, M. G., et al. J Clin Invest, 1999. 104 (6): p. 777-85,
Chiaramonte, M. G., et al. Hepatology, 2001. 34(2): p.
273-82.].
[0026] In another embodiment, the disorder or condition to be
treated is Hodgkin's disease. Hodgkin's disease is unusual among
malignancies in that the neoplastic Reed-Sternberg cell, often
derived from B-cells, make up only a small proportion of the
clinically detectable mass. Hodgkin's disease-derived cell lines
and primary ReedSternberg cells frequently express IL-13 and its
receptor [Skinnider, B. F., et al. Blood, 2001. 97(1): p. 250-5].
As IL-13 promotes cell survival and proliferation in normal
B-cells, it was proposed that IL-13 could act as a growth factor
for Reed-Sternberg cells. Skinnider et al. have demonstrated that
neutralising antibodies against IL-13 can inhibit the growth of
Hodgkin's disease-derived cell lines in vitro [Kapp, U., et al. J
Exp Med, 1999. 189 (12): p. 1939-46.]. This finding suggested that
Reed-Sternberg cells might enhance their own survival by an IL-13
autocrine and paracrine cytokine loop. Consistent with this
hypothesis, raised levels of IL-13 have been detected in the serum
of some Hodgkin's disease patients when compared to normal controls
[Fiumara, P., F. Cabanillas, and A. Younes, Blood, 2001. 98 (9): p.
2877-8.]. IL-13 inhibitors may therefore prevent disease
progression by inhibiting proliferation of malignant Reed-Sternberg
cells.
[0027] In another embodiment, the disorder or condition to be
treated is tumour recurrence or metastasis. Inhibition of IL-13 has
been shown to enhance anti-viral vaccines in animal models and may
be beneficial in the treatment of HIV and other infectious diseases
[Ahlers, J. D., et al. Proc Natl Acad Sci USA, 2002]. Many human
cancer cells express immunogenic tumour specific antigens. However,
although many tumours spontaneously regress, a number evade the
immune system (immunosurveillance) by suppressing T-cell mediated
immunity. Terabe et al. Nat Immunol, 2000. 1 (6): p. 515-20, have
demonstrated a role of IL-13 in immunosuppression in a mouse model
in which tumours spontaneously regress after initial growth and
then recur. Specific inhibition of IL-13, with soluble IL-13Ra2,
protected these mice from tumour recurrence. Terabe et al went on
to show that IL-13 suppresses the differentiation of tumour
specific CD8+ cytotoxic lymphocytes that mediate anti-tumour immune
responses.
[0028] In another embodiment, the disorder or condition to be
treated is a respiratory viral infection, which exacerbates
underlying chronic conditions such as asthma, chronic bronchitis,
COPD, otitis media, and sinusitis. The respiratory viral infection
treated may be associated with secondary bacterial infection, such
as otitis media, sinusitis or pneumonia.
[0029] In another embodiment, the disorder or condition to be
treated is selected from other diseases or conditions, in
particular diseases or conditions having an inflammatory component,
for example, diseases of the bone and joints including rheumatoid
arthritis, psoriatic arthritis, and other diseases such as
atherosclerosis, multiple sclerosis, and acute and chronic
allograft rejection, e.g. following transplantation of heart,
kidney, liver, lung or bone marrow.
[0030] In another embodiment, the disorder or condition to be
treated is endotoxic shock, glomerulonephritis, cerebral and
cardiac ischemia, Alzheimer's disease, cystic fibrosis, virus
infections and the exacerbations associated with them, acquired
immune deficiency syndrome (AIDS), multiple sclerosis (MS),
Helicobacter pylori associated gastritis, and cancers, particularly
the growth of ovarian cancer.
[0031] In another embodiment, the disorder or condition to be
treated is the symptoms caused by viral infection in a human which
is caused by the human rhinovirus, other enterovirus, coronavirus,
herpes viruses, influenza virus, parainfluenza virus, respiratory
syncytial virus or an adenovirus.
[0032] Treatment in accordance with the present invention may be
symptomatic or prophylactic.
[0033] The effectiveness of an agent of the invention in inhibiting
inflammatory conditions, for example in inflammatory airways
diseases, may be demonstrated in an animal model, e.g. mouse, rat
or rabbit model, of airway inflammation or other inflammatory
conditions, for example as described by Wada et al, J. Exp. Med
(1994) 180:1135-40; Sekido et al, Nature (1993) 365:654-57;
Modelska et al., Am. J. Respir. Crit. Care. Med (1999) 160:1450-56;
and Laffon et al (1999) Am. J. Respir. Crit. Care Med.
160:1443-49.
[0034] In yet another embodiment, the invention provides a method
for identifying a cell having a receptor for IL-13. This method
involves contacting the cell with any of the above antibodies or
antibody fragments further having a detectable label. The label is
radioactive, fluorescent, magnetic, paramagnetic, or
chemiluminescent. The method further can involve any of the above
imaging or separating the labeled cell.
[0035] In another embodiment, any of the above human or humanized
antibodies or antibody fragments are synthetic.
[0036] In another embodiment, the invention provides a
pharmaceutical composition and an additional therapeutic agent.
[0037] The additional therapeutic agent can be selected from the
group consisting of anti-inflammatory, bronchodilatory,
antihistamine or anti-tussive drug substances, particularly in the
treatment of obstructive or inflammatory airways diseases such as
those mentioned hereinbefore, for example as potentiators of
therapeutic activity of such drugs or as a means of reducing
required dosaging or potential side effects of such drugs. A
therapeutic agent of the invention may be mixed with the other drug
substance in a fixed pharmaceutical composition or it may be
administered separately, before, simultaneously with or after the
other drug substance. Accordingly the invention includes a
combination of an agent of the invention as hereinbefore described
with an anti-inflammatory, bronchodilatory, antihistamine or
anti-tussive drug substance, said agent of the invention and said
drug substance being in the same or different pharmaceutical
composition.
[0038] Suitable anti-inflammatory drugs include steroids, in
particular glucocorticosteroids such as budesonide, beclamethasone
dipropionate, fluticasone propionate, ciclesonide or mometasone
furoate, or steroids described in WO 02/88167, WO 02/12266, WO
02/100879, WO 02/00679 (especially those of Examples 3, 11, 14, 17,
19, 26, 34, 37, 39, 51, 60, 67, 72, 73, 90, 99 and 101), WO
03/35668, WO 03/48181, WO 03/62259, WO 03/64445, WO 03/72592, WO
04/39827 and WO 04/66920; non-steroidal glucocorticoid receptor
agonists, such as those described in DE 10261874, WO 00/00531, WO
02/10143, WO 03/82280, WO 03/82787, WO 03/86294, WO 03/104195, WO
03/101932, WO 04/05229, WO 04/18429, WO 04/19935 and WO 04/26248;
LTB4 antagonists such as BIIL 284, CP-195543, DPC11870, LTB4
ethanolamide, LY 293111, LY 255283, CGS025019C, CP-195543,
ONO-4057, SB 209247, SC-53228 and those described in U.S. Pat. No.
5,451,700; LTD4 antagonists such include montelukast, pranlukast,
zafirlukast, accolate, SR2640, Wy-48,252, ICI 198615, MK-571,
LY-171883, Ro 24-5913 and L-648051; PDE4 inhibitors such cilomilast
(Ariflo.RTM. GlaxoSmithKline), Roflumilast (Byk Gulden), V-11294A
(Napp), BAY19-8004 (Bayer), SCH-351591 (Schering-Plough),
Arofylline (Almirall Prodesfarma), PD189659/PD168787 (Parke-Davis),
AWD-12-281 (Asta Medica), CDC-801 (Celgene), SelCID.TM. CC-10004
(Celgene), VM554/UM565 (Vernalis), T-440 (Tanabe), KW-4490 (Kyowa
Hakko Kogyo), and those disclosed in WO 92/19594, WO 93/19749, WO
93/19750, WO 93/19751, WO 98/18796, WO 99/16766, WO 01/13953, WO
03/104204, WO 03/104205, WO 03/39544, WO 04/000814, WO 04/000839,
WO 04/005258, WO 04/018450, WO 04/018451, WO 04/018457, WO
04/018465, WO 04/018431, WO 04/018449, WO 04/018450, WO 04/018451,
WO 04/018457, WO 04/018465, WO 04/019944, WO 04/019945, WO
04/045607 and WO 04/037805; A.sub.2A agonists such as those
described in EP 1052264, EP 1241176, EP 409595A2, WO 94/17090, WO
96/02543, WO 96/02553, WO 98/28319, WO 99/24449, WO 99/24450, WO
99/24451, WO 99/38877, WO 99/41267, WO 99/67263, WO 99/67264, WO
99/67265, WO 99/67266, WO 00/23457, WO 00/77018, WO 00/78774, WO
01/23399, WO 01/27130, WO 01/27131, WO 01/60835, WO 01/94368, WO
02/00676, WO 02/22630, WO 02/96462, and WO 03/086408; and A.sub.2B
antagonists such as those described in WO 02/42298.
[0039] Suitable bronchodilatory drugs include anticholinergic or
antimuscarinic agents, in particular ipratropium bromide,
oxitropium bromide, tiotropium salts and CHF 4226 (Chiesi), and
glycopyrrolate, but also those described in EP 424021, U.S. Pat.
No. 3,714,357, U.S. Pat. No. 5,171,744, WO 01/04118, WO 02/00652,
WO 02/51841, WO 02/53564, WO 03/00840, WO 03/33495, WO 03/53966, WO
03/87094, WO 04/018422 and WO 04/05285; and beta-2 adrenoceptor
agonists such as albuterol (salbutamol), metaproterenol,
terbutaline, salmeterol fenoterol, procaterol, and especially,
formoterol, carmoterol and pharmaceutically acceptable salts
thereof, and compounds (in free or salt or solvate form) of formula
I of WO 00/75114, which document is incorporated herein by
reference, preferably compounds of the Examples thereof, especially
a compound of formula
##STR00001##
i.e.,
(5-[(R)-2-(5,6-Diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy--
1H-quinolin-2-one) and pharmaceutically acceptable salts thereof,
as well as compounds (in free or salt or solvate form) of formula I
of WO 04/16601, and also compounds of EP 1440966, JP 05025045, WO
93/18007, WO 99/64035, US 2002/0055651, WO 01/42193, WO 01/83462,
WO 02/66422, WO 02/70490, WO 02/76933, WO 03/24439, WO 03/42160, WO
03/42164, WO 03/72539, WO 03/91204, WO 03/99764, WO 04/16578, WO
04/22547, WO 04/32921, WO 04/33412, WO 04/37768, WO 04/37773, WO
04/37807, WO 04/39762, WO 04/39766, WO 04/45618 WO 04/46083, WO
04/80964, EP1460064, WO 04/087142, WO 04/089892, EP 01477167, US
2004/0242622, US 2004/0229904, WO 04/108675, WO 04/108676, WO
05/033121, WO 05/040103 and WO 05/044787.
[0040] Suitable dual anti-inflammatory and bronchodilatory drugs
include dual beta-2 adrenoceptor agonist/muscarinic antagonists
such as those disclosed in US 2004/0167167, WO 04/74246 and WO
04/74812.
[0041] Suitable antihistamine drug substances include cetirizine
hydrochloride, acetaminophen, clemastine fumarate, promethazine,
loratidine, desloratidine, diphenhydramine and fexofenadine
hydrochloride, activastine, astemizole, azelastine, ebastine,
epinastine, mizolastine and tefenadine as well as those disclosed
in JP 2004107299, WO 03/099807 and WO 04/026841.
[0042] Combinations of therapeutic agents of the invention and
anticholinergic or antimuscarinic agents, steroids, beta-2
agonists, PDE4 inhibitors, dopamine receptor agonists, LTD4
antagonists or LTB4 antagonists may also be used. Other useful
combinations of agents of the invention with anti-inflammatory
drugs are those with other antagonists of chemokine receptors, e.g.
CCR-1, CCR-3, CCR-4, CCR-5, CCR-6, CCR-7, CCR-8, CCR-9 and CCR10,
CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, particularly CCR-5 antagonists
such as Schering-Plough antagonists SC-351125, SCH-55700 and SCH-D,
Takeda antagonists such as
N-[[4-[[[6,7-dihydro-2-(4-methylphenyl)-5H-benzocyclohepten-8-yl]carbonyl-
]amino]phenyl]-methyl]-tetrahydro-N,N-dimethyl-2H-pyran-4-aminium
chloride (TAK-770), CCR-5 antagonists described in U.S. Pat. No.
6,166,037 (particularly claims 18 and 19), WO 0066558 (particularly
claim 8), WO 0066559 (particularly claim 9), WO 04/018425 and WO
04/026873.
[0043] The additional therapeutic agent may also be selected from
the group consisting of other cytokine binding molecules,
particularly antibodies of other cytokines, in particular a
combination with an anti-IL-4 antibody, such as described in
PCT/EP2005/00836, an anti-IgE antibody, such as Xolair.RTM., an
anti-IL31 antibody, an anti-IL31R antibody, an anti-TSLP antibody,
an anti-TSLP receptor antibody, an anti-endoglin antibody, an
anti-IL1b antibody or another anti-IL13 antibody, such as described
in WO05/007699.
[0044] In a certain embodiment, the invention provides an antibody
having a first amino acid sequence which is a heavy chain selected
from one to three of SEQ ID NOs: 6-10, and a sequence having at
least 60, 70, 80, 90 or 95 percent sequence identity in the CDR
regions with the CDR regions having SEQ ID NOs: 6-10; and a second
amino acid sequence which is a light chain selected from one to
three of SEQ ID NOs: 16-22, and a sequence having at least 60, 70,
80, 90 or 95 percent sequence identity in the CDR regions with the
CDR regions shown in SEQ ID NOs: 16-22.
[0045] In still another embodiment, the invention provides an
immunoconjugate made out of a first component which is an antibody
or fragment thereof and a second component having a second amino
acid sequence. For example, the immunoconjugate is a cytotoxin, or
the immunoconjugate is a binding protein or antibody having a
binding specificity for a target that is different from IL-13.
[0046] In certain embodiments, the invention provides for a
bispecific antibody.
[0047] In another embodiment, the invention provides a kit having
an antibody or antibody fragment thereof. In some embodiments, the
kit further contains a pharmaceutically acceptable carrier or
excipient therefore. In other related embodiments, the antibody in
the kit is present in a unit dose. In yet another related
embodiment, the kit includes instructions for use in administering
to a subject.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The present invention relates to isolated antibodies,
particularly human antibodies, that bind specifically to IL-13 and
that inhibit functional properties of IL-13. In certain
embodiments, the antibodies of the invention are derived from
particular heavy and light chain sequences and/or comprise
particular structural features such as CDR regions comprising
particular amino acid sequences. The invention provides isolated
antibodies, methods of making such antibodies, immunoconjugates and
bispecific molecules comprising such antibodies and pharmaceutical
compositions containing the antibodies, immunoconjugates or
bispecific molecules of the invention. The invention also relates
to methods of using the antibodies to inhibit a disorder or
condition associated with the presence of cell receptor target
IL-13, for example, in the treatment of an inflammatory or allergic
condition, particularly an inflammatory or obstructive airways
disease.
[0049] In order that the present invention may be more readily
understood, certain terms are first defined. Additional definitions
are set forth throughout the detailed description.
[0050] The term `interleukin-13` or `IL-13` is, except where
context dictates otherwise, reference to human IL-13. The present
invention provides antibodies to human IL-13, especially human
antibodies, that are cross-reactive with non-human primate IL-13,
including cynomolgus and rhesus monkey IL-13. Antibodies in
accordance with some embodiments of the present invention recognise
a variant of IL-13 in which the arginine residue at amino acid
position 130 is replaced by glutamine. In other aspects and
embodiments the present invention provides specific binding members
against murine IL-13, specifically mouse IL-13.
[0051] The term "immune response" refers to the action of, for
example, lymphocytes, antigen presenting cells, phagocytic cells,
granulocytes, and soluble macromolecules produced by the above
cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or
elimination from the human body of invading pathogens, cells or
tissues infected with pathogens, cancerous cells, or, in cases of
autoimmunity or pathological inflammation, normal human cells or
tissues.
[0052] A "signal transduction pathway" refers to the biochemical
relationship between a variety of signal transduction molecules
that play a role in the transmission of a signal from one portion
of a cell to another portion of a cell. As used herein, the phrase
"cell surface receptor" includes, for example, molecules and
complexes of molecules capable of receiving a signal and capable of
the transmission of such a signal across the plasma membrane of a
cell. An example of a "cell surface receptor" of the present
invention is the IL-13 receptor to which the IL-13 protein molecule
binds.
[0053] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chains thereof. A naturally occurring
"antibody" is a glycoprotein comprising at least two heavy (H)
chains and two light (L) chains inter-connected by disulfide bonds.
Each heavy chain is comprised of a heavy chain variable region
(abbreviated herein as V.sub.H) and a heavy chain constant region.
The heavy chain constant region is comprised of three domains, CH1,
CH2 and CH3. Each light chain is comprised of a light chain
variable region (abbreviated herein as V.sub.L) and a light chain
constant region. The light chain constant region is comprised of
one domain, C.sub.L. The V.sub.H and V.sub.L regions can be further
subdivided into regions of hypervariability, termed complementarity
determining regions (CDR), interspersed with regions that are more
conserved, termed framework regions (FR). Each V.sub.H and V.sub.L
is, composed of three CDRs and four FRs arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy
and light chains contain a binding domain that interacts with an
antigen. The constant regions of the antibodies may mediate the
binding of the immunoglobulin to host tissues or factors, including
various cells of the immune system (e.g., effector cells) and the
first component (C1q) of the classical complement system.
[0054] The term "antigen-binding portion" of an antibody (or simply
"antigen portion"), as used herein, refers to one or more fragments
of an antibody that retain the ability to specifically bind to an
antigen (e.g., IL-13). It has been shown that the antigen-binding
function of an antibody can be performed by fragments of a
full-length antibody. Examples of binding fragments encompassed
within the term "antigen-binding portion" of an antibody include a
Fab fragment, a monovalent fragment consisting of the V.sub.L,
V.sub.H, C.sub.L and CH1 domains; a F(ab).sub.2 fragment, a
bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; a Fd fragment consisting of
the V.sub.H and CH1 domains; a Fv fragment consisting of the
V.sub.L and V.sub.H domains of a single arm of an antibody; a dAb
fragment (Ward et al., 1989 Nature 341:544-546), which consists of
a V.sub.H domain; and an isolated complementarity determining
region (CDR).
[0055] Furthermore, although the two domains of the Fv fragment,
V.sub.L and V.sub.H, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the
V.sub.L and V.sub.H regions pair to form monovalent molecules
(known as single chain Fv (scFv); see e.g., Bird et al., 1988
Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci.
85:5879-5883). Such single chain antibodies are also intended to be
encompassed within the term "antigen-binding portion" of an
antibody. These antibody fragments are obtained using conventional
techniques known to those of skill in the art, and the fragments
are screened for utility in the same manner as are intact
antibodies.
[0056] An "isolated antibody", as used herein, refers to an
antibody that is substantially free of other antibodies having
different antigenic specificities (e.g., an isolated antibody that
specifically binds IL-13 is substantially free of antibodies that
specifically bind antigens other than IL-13). An isolated antibody
that specifically binds IL-13 may, however, have cross-reactivity
to other antigens, such as IL-13 molecules from other species.
Moreover, an isolated antibody may be substantially free of other
cellular material and/or chemicals.
[0057] The terms "monoclonal antibody" or "monoclonal antibody
composition" as used herein refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope.
[0058] The term "human antibody", as used herein, is intended to
include antibodies having variable regions in which both the
framework and CDR regions are derived from sequences of human
origin. Furthermore, if the antibody contains a constant region,
the constant region also is derived from such human sequences,
e.g., human germline sequences, or mutated versions of human
germline sequences. The human antibodies of the invention may
include amino acid residues not encoded by human sequences (e.g.,
mutations introduced by random or site-specific mutagenesis in
vitro or by somatic mutation in vivo). However, the term "human
antibody", as used herein, is not intended to include antibodies in
which CDR sequences derived from the germline of another mammalian
species, such as a mouse, have been grafted onto human framework
sequences.
[0059] The term "human monoclonal antibody" refers to antibodies
displaying a single binding specificity which have variable regions
in which both the framework and CDR regions are derived from human
sequences. In one embodiment, the human monoclonal antibodies are
produced by a hybridoma which includes a B cell obtained from a
transgenic nonhuman animal, e.g., a transgenic mouse, having a
genome comprising a human heavy chain transgene and a light chain
transgene fused to an immortalized cell.
[0060] The term "recombinant human antibody", as used herein,
includes all human antibodies that are prepared, expressed, created
or isolated by recombinant means, such as antibodies isolated from
an animal (e.g., a mouse) that is transgenic or transchromosomal
for human immunoglobulin genes or a hybridoma prepared therefrom,
antibodies isolated from a host cell transformed to express the
human antibody, e.g., from a transfectoma, antibodies isolated from
a recombinant, combinatorial human antibody library, and antibodies
prepared, expressed, created or isolated by any other means that
involve splicing of all or a portion of a human immunoglobulin
gene, sequences to other DNA sequences. Such recombinant human
antibodies have variable regions in which the framework and CDR
regions are derived from human germline immunoglobulin sequences.
In certain embodiments, however, such recombinant human antibodies
can be subjected to in vitro mutagenesis (or, when an animal
transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the V.sub.H and
V.sub.L regions of the recombinant antibodies are sequences that,
while derived from and related to human germline V.sub.H and
V.sub.L sequences, may not naturally exist within the human
antibody germline repertoire in vivo.
[0061] As used herein, "isotype" refers to the antibody class
(e.g., IgM, IgE, IgG such as IgG1 or IgG4) that is encoded by the
heavy chain constant region genes.
[0062] The phrases "an antibody recognizing an antigen" and "an
antibody specific for an antigen" are used interchangeably herein
with the term "an antibody which binds specifically to an
antigen."
[0063] As used herein, an antibody that "specifically binds to
human IL-13" is intended to refer to an antibody that binds to
human IL-13 with a K.sub.D of 5.times.10.sup.-9 M or less. An
antibody that "cross-reacts with an antigen other than human IL-13"
is intended to refer to an antibody that binds that antigen with a
5.times.10.sup.-9 M or less. An antibody that "does not cross-react
with a particular antigen" is intended to refer to an antibody that
binds to that antigen, with a K.sub.D of 1.5.times.10.sup.-8 M or
greater, or a K.sub.D of 5-10.times.10.sup.-8 M or
1.times.10.sup.-7 M or greater. In certain embodiments, such
antibodies that do not cross-react with the antigen exhibit
essentially undetectable binding against these proteins in standard
binding assays.
[0064] As used herein, an antibody that "inhibits binding of IL-13
to the IL-13 receptor" refers to an antibody that inhibits IL-13
binding to the receptor with a K.sub.D of 5 nM or less.
[0065] As used herein, an antibody that "inhibits inflammatory
mediator release" is intended to refer to an antibody that inhibits
IL-13 induced eotaxin release from human lung fibroblasts with an
IC.sub.50 less than 10 nM, 5 nM, 2.5 nM, 1.0 nM, 0.5 nM, or
less.
[0066] The term "K.sub.assoc" or "K.sub.a", as used herein, is
intended to refer to the association rate of a particular
antibody-antigen interaction, whereas the term "K.sub.dis" or
"K.sub.D," as used herein, is intended to refer to the dissociation
rate of a particular antibody-antigen interaction. The term
"K.sub.D", as used herein, is intended to refer to the dissociation
constant, which is obtained from the ratio of K.sub.d to K.sub.a
(i.e. K.sub.d/K.sub.a) and is expressed as a molar concentration
(M). K.sub.D values for antibodies can be determined using methods
well established in the art. A method for determining the K.sub.D
of an antibody is by using surface plasmon resonance, or using a
biosensor system such as a Biacore.RTM. system.
[0067] As used herein, the term "high affinity" for an IgG antibody
refers to an antibody having a K.sub.D of 10.sup.-8 M or less,
10.sup.-9 M or less, or 10.sup.-10 M or less for a target
antigen.
[0068] As used herein, the term "subject" includes any human or
nonhuman animal. The term "nonhuman animal" includes all
vertebrates, e.g., mammals and non-mammals, such as nonhuman
primates, sheep, dogs, cats, horses, cows chickens, amphibians,
reptiles, etc.
[0069] Various aspects of the invention are described in further
detail in the following subsections.
[0070] Standard assays to evaluate the binding ability of the
antibodies toward IL-13 of various species are known in the art,
including for example, ELISAs, western blots and RIAs. Suitable
assays are described in detail in the Examples. The binding
kinetics (e.g., binding affinity) of the antibodies also can be
assessed by standard assays known in the art, such as by Biacore
analysis. Assays to evaluate the effects of the antibodies on
functional properties of IL-13 are described in further detail in
the Examples.
[0071] Accordingly, an antibody that "inhibits" one or more of
these IL-13 functional properties (e.g., biochemical,
immunochemical, cellular, physiological or other biological
activities, or the like) as determined according to methodologies
known to the art and described herein, will be understood to relate
to a statistically significant decrease in the particular activity
relative to that seen in the absence of the antibody (e.g., or when
a control antibody of irrelevant specificity is present). An
antibody that inhibits IL-13 activity effects such a statistically
significant decrease by at least 10% of the measured parameter, by
at least 50%, 80% or 90%, and in certain embodiments an antibody of
the invention may inhibit greater than 95%, 98% or 99% of IL-13
functional activity.
Monoclonal Antibodies
[0072] Antibodies of the invention are the human monoclonal
antibodies, isolated and structurally characterized as described,
in Examples 1-5. The V.sub.H amino acid sequences of the antibodies
are shown in SEQ ID NOs: 6-10 respectively. The V.sub.L amino acid
sequences of the antibodies are shown in SEQ ID NOs: 16-22
respectively. Other antibodies of the invention include amino acids
that have been mutated, yet have at least 60, 70, 80, 90 or 95
percent identity in the CDR regions with the CDR regions depicted
in the sequences described above.
[0073] Since each of these antibodies can bind to IL-13, the
V.sub.H and V.sub.L sequences can be "mixed and matched" to create
other anti-IL-13 binding molecules of the invention. IL-13 binding
of such "mixed and matched" antibodies can be tested using the
binding assays described above and in the Examples (e.g., ELISAs).
When V.sub.H and V.sub.L chains are mixed and matched, a V.sub.H
sequence from a particular V.sub.H/V.sub.L pairing should be
replaced with a structurally similar V.sub.H sequence. Likewise, a
V.sub.L sequence from a particular V.sub.H/V.sub.L pairing should
be replaced with a structurally similar V.sub.L sequence. The
V.sub.H and V.sub.L sequences of the antibodies of the present
invention are particularly amenable for mixing and matching, since
these antibodies use V.sub.H and V.sub.L sequences derived from the
same germline sequences and thus exhibit structural similarity.
[0074] In another aspect, the invention provides antibodies that
comprise the heavy chain and light chain CDR1s, CDR2s and CDR3s of
the antibodies, or combinations thereof. The amino acid sequences
of the V.sub.H CDR1s of the antibodies are shown in SEQ ID NOs:
6-7. The amino acid sequence of the V.sub.H CDR2s of the antibodies
is shown by SEQ ID NO: 8. The amino acid sequences of the V.sub.H
CDR3s of the antibodies are shown in SEQ ID NOs: 9-10. The amino
acid sequences of the V.sub.L CDR1s of the antibodies are shown in
SEQ ID NOs: 16-18. The amino acid sequences of the V.sub.L CDR2s of
the antibodies is shown in SEQ ID NO: 19. The amino acid sequences
of the V.sub.L CDR3s of the antibodies are shown in SEQ ID NOs:
20-22. The CDR regions are delineated using the Kabat system
(Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242).
[0075] Given that each of these antibodies can bind to IL-13 and
that antigen-binding specificity is provided primarily by the CDR1,
2 and 3 regions, the V.sub.H CDR1, 2 and 3 sequences and V.sub.L
CDR1, 2 and 3 sequences can be "mixed and matched" (i.e., CDRs from
different antibodies can be mixed and match, although each antibody
must contain a V.sub.H CDR1, 2 and 3 and a V.sub.L CDR1, 2 and 3)
to create other anti-IL-13 binding molecules of the invention.
IL-13 binding of such "mixed and matched" antibodies can be tested
using the binding assays described above and in the Examples (e.g.,
ELISAs). When V.sub.H CDR sequences are mixed and matched, the
CDR1, CDR2 and/or CDR3 sequence from a particular V.sub.H sequence
should be replaced with a structurally similar CDR sequence(s).
Likewise, when V.sub.L CDR sequences are mixed and matched, the
CDR1, CDR2 and/or CDR3 sequence from a particular V.sub.L sequence
should be replaced with a structurally similar CDR sequence(s). It
will be readily apparent to the ordinarily skilled artisan that
novel V.sub.H and V.sub.L sequences can be created by substituting
one or more V.sub.H and/or V.sub.L CDR region sequences with
structurally similar sequences from the CDR sequences shown herein
for monoclonal antibodies of the present invention.
[0076] An isolated monoclonal antibody, or antigen binding portion
thereof has: a heavy chain variable region CDR1 comprising an amino
acid sequence selected from the group consisting of SEQ ID NOs:
6-7; a heavy chain variable region CDR2 comprising an amino acid
sequence of SEQ ID NO: 8; a heavy chain variable region CDR3
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 9-10; a light chain variable region CDR1
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 16-18; a light chain variable region CDR2
comprising an amino acid sequence of SEQ ID NO: 19; and a light
chain variable region CDR3 comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 20-22; wherein
the antibody specifically binds IL-13.
[0077] In a certain embodiment, the antibody consists of: a heavy
chain variable region CDR1 comprising SEQ ID NO: 6; a heavy chain
variable region CDR2 comprising SEQ ID NO: 8; a heavy chain
variable region CDR3 comprising SEQ ID NO: 9; a light chain
variable region CDR1 comprising SEQ ID NO: 16; a light chain
variable region CDR2 comprising SEQ ID NO: 19; and a light chain
variable region CDR3 comprising SEQ ID NO: 20.
[0078] In another embodiment, the antibody consists of: a heavy
chain variable region CDR1 comprising SEQ ID NO: 7; a heavy chain
variable region CDR2 comprising SEQ ID NO: 8; a heavy chain
variable region CDR3 comprising SEQ ID NO: 10; a light chain
variable region CDR1 comprising SEQ ID NO: 17; a light chain
variable region CDR2 comprising SEQ ID NO: 19; and a light chain
variable region CDR3 comprising SEQ ID NO: 21.
[0079] In yet another embodiment, the antibody consists of: a heavy
chain variable region CDR1 comprising SEQ ID NO: 7; a heavy chain
variable region CDR2 comprising SEQ ID NO: 8; a heavy chain
variable region CDR3 comprising SEQ ID NO: 10; a light chain
variable region CDR1 comprising SEQ ID NO: 18; a light chain
variable region CDR2 comprising SEQ ID NO: 19; and a light chain
variable region CDR3 comprising SEQ ID NO: 22.
[0080] As used herein, a human antibody comprises heavy or light
chain variable regions that is "the product of" or "derived from" a
particular germline sequence if the variable regions of the
antibody are obtained from a system that uses human germline
immunoglobulin genes.
[0081] Such systems include immunizing a transgenic mouse carrying
human immunoglobulin genes with the antigen of interest or
screening a human immunoglobulin gene library displayed on phage
with the antigen of interest. A human antibody that is "the product
of" or "derived from" a human germline immunoglobulin sequence can
be identified as such by comparing the amino acid sequence of the
human antibody to the amino acid sequences of human germline
immunoglobulins and selecting the human germline immunoglobulin
sequence that is closest in sequence (i.e., greatest % identity) to
the sequence of the human antibody. A human antibody that is "the
product of" or "derived from" a particular human germline
immunoglobulin sequence may contain amino acid differences as
compared to the germline sequence, due to, for example, naturally
occurring somatic mutations or intentional introduction of
site-directed mutation. However, a selected human antibody
typically is at least 90% identical in amino acids sequence to an
amino acid sequence encoded by a human germline immunoglobulin gene
and contains amino acid residues that identify the human antibody
as being human when compared to the germline immunoglobulin amino
acid sequences of other species (e.g., murine germline sequences).
In certain cases, a human antibody may be at least 60%, 70%, 80%,
90%, or at least 95%, or even at least 96%, 97%, 98%, or 99%
identical in amino acid sequence to the amino acid sequence encoded
by the germline immunoglobulin gene. Typically, a human antibody
derived from a particular human germline sequence will display no
more than 10 amino acid differences from the amino acid sequence
encoded by the human germline immunoglobulin gene. In certain
cases, the human antibody may display no more than 5, or even no
more than 4, 3, 2, or 1 amino acid difference from the amino acid
sequence encoded by the germline immunoglobulin gene.
Homologous Antibodies
[0082] In yet another embodiment, an antibody of the invention has
heavy and light chain variable regions having amino acid sequences
that are homologous to the amino acid sequences of the antibodies
described herein, and wherein the antibodies retain the desired
functional properties of the anti-IL-13 antibodies of the
Invention.
[0083] For example, the invention provides an isolated monoclonal
antibody, or antigen binding portion thereof, comprising a heavy
chain variable region and a light chain variable region, wherein:
the heavy chain variable region comprises an amino acid sequence
that is at least 80% homologous to an amino acid sequence selected
from the group consisting of SEQ ID NOs: 6-10; the light chain
variable region comprises an amino acid sequence that is at least
80% homologous to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 16-22; the antibody specifically binds to
IL-13, and the antibody exhibits at least one of the following
functional properties: the antibody inhibits binding IL-13 protein
to the IL-13 receptor or the antibody inhibits IL-13 receptor
binding preventing or ameliorating an inflammatory or allergic
condition, particularly an inflammatory or obstructive airways
disease, or the antibody inhibits IL-13 receptor binding preventing
or ameliorating asthma or the antibody inhibits IL-13 receptor
binding preventing or ameliorating COPD.
[0084] In various embodiments, the antibody may exhibit one or
more, two or more, or three of the functional properties discussed
above. The antibody can be, for example, a human antibody, a
humanized antibody or a chimeric antibody.
[0085] In other embodiments, the V.sub.H and/or V.sub.L amino acid
sequences may be 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%
homologous to the sequences set forth above. An antibody having
V.sub.H and V.sub.L regions having high (i.e., 80% or greater)
homology to the V.sub.H and V.sub.L regions of SEQ ID NOs: 6-10 and
16-22 respectively, can be obtained by mutagenesis (e.g.,
site-directed or PCR-mediated mutagenesis) of nucleic acid
molecules encoding SEQ ID NOs: 6-10 and/or 16-22, followed by
testing of the encoded altered antibody for retained function
(i.e., the functions set forth above) using the functional assays
described herein.
[0086] As used herein, the percent homology between two amino acid
sequences is equivalent to the percent identity between the two
sequences. The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences (i.e., % homology=# of identical positions/total # of
positions.times.100), taking into account the number of gaps, and
the length of each gap, which need to be introduced for optimal
alignment of the two sequences. The comparison of sequences and
determination of percent identity between two sequences can be
accomplished using a mathematical algorithm, as described in the
non-limiting examples below.
[0087] The percent identity between two amino acid sequences can be
determined using the algorithm of E. Meyers and W. Miller (Comput.
Appl. Biosci., 4:11-17, 1988) which has been incorporated into the
ALIGN program (version 2.0), using a PAM120 weight residue table, a
gap length penalty of 12 and a gap penalty of 4. In addition, the
percent identity between two amino acid sequences can be determined
using the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970)
algorithm which has been incorporated into the GAP program in the
GCG software package (available at http://www.gcg.com), using
either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of
16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or
6.
[0088] Additionally or alternatively, the protein sequences of the
present invention can further be used as a "query sequence" to
perform a search against public databases to, for example, identify
related sequences. Such searches can be performed using the XBLAST
program (version 2.0) of Altschul, et al., 1990 J. Mol. Biol.
215:403-10. BLAST protein searches can be performed with the XBLAST
program, score=50, wordlength=3 to obtain amino acid sequences
homologous to the antibody molecules of the invention. To obtain
gapped alignments for comparison purposes, Gapped BLAST can be
utilized as described in Altschul et al., 1997 Nucleic Acids Res.
25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and
NBLAST) can be used. See http:Ilwww.ncbi.nhn.nih.gov.
Antibodies with Conservative Modifications
[0089] In certain embodiments, an antibody of the invention has a
heavy chain variable region consist of CDR1, CDR2, and CDR3
sequences and a light chain variable region consisting of CDR1,
CDR2, and CDR3 sequences, wherein one or more of these CDR
sequences have specified amino acid sequences based on the
antibodies described herein or conservative modifications thereof,
and wherein the antibodies retain the desired functional properties
of the anti-IL-13 antibodies of the invention. Accordingly, the
invention provides an isolated monoclonal antibody, or antigen
binding portion thereof, consisting of a heavy chain variable
region consisting of CDR1, CDR2, and CDR3 sequences and a light
chain variable region consisting of CDR1, CDR2, and CDR3 sequences,
wherein: the heavy chain variable regions of CDR1 is sequences
consisting of amino acid sequences selected from the group
consisting of amino acid sequences of SEQ ID NOs: 6-7, and
conservative modifications thereof, the heavy chain variable region
of CDR2 is a sequence consisting of an amino acid sequence of SEQ
ID NO: 8, and conservative modifications thereof, the heavy chain
variable region of CDR3 is sequences consisting of amino acid
sequences selected from the group consisting of amino acid
sequences of SEQ ID NOs: 9-10, and conservative modifications
thereof, the light chain variable regions of CDR1 is sequences
consisting of amino acid sequences selected from the group
consisting of amino acid sequences of SEQ ID NOs: 16-18, and
conservative modifications thereof; the light chain variable
regions of CDR2 is a sequence consisting of an amino acid sequence
of SEQ ID NO: 19, and conservative modifications thereof; the light
chain variable regions of CDR3 is sequences consisting of amino
acid sequences selected from the group consisting of amino acid
sequences of SEQ ID NOs: 20-22, and conservative modifications
thereof; the antibody specifically binds to IL-13; and the antibody
inhibits IL-13 receptor binding preventing inflammatory mediator
release.
[0090] In various embodiments, the antibody may exhibit one or
more, two or more, or three or more of the functional properties
listed discussed above. Such antibodies can be, for example, human
antibodies, humanized antibodies or chimeric antibodies.
[0091] As used herein, the term "conservative sequence
modifications" is intended to refer to amino acid modifications
that do not significantly affect or alter the binding
characteristics of the antibody containing the amino acid sequence.
Such conservative modifications include amino acid substitutions,
additions and deletions. Modifications can be introduced into an
antibody of the invention by standard techniques known in the art,
such as site-directed mutagenesis and PCR-mediated mutagenesis.
[0092] Conservative amino acid substitutions are ones in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine, tryptophan),
nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,
proline, phenylalanine, methionine), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one
or more amino acid residues within the CDR regions of an antibody
of the invention can be replaced with other amino acid residues
from the same side chain family, and the altered antibody can be
tested for retained function using the functional assays described
herein.
Antibodies that Bind to the Same Epitope as Anti-IL-13 Antibodies
of the Invention
[0093] In another embodiment, the invention provides antibodies
that bind to the same epitope as do the various anti-IL-13
antibodies of the invention provided herein. Such additional
antibodies can be identified based on their ability to
cross-compete (e.g., to competitively inhibit the binding of, in a
statistically significant manner) with other antibodies of the
invention in standard IL-13 binding assays. The ability of a test
antibody to inhibit the binding of antibodies of the present
invention to human IL-13 demonstrates that the test antibody can
compete with that antibody for binding to human IL-13; such an
antibody may, according to non-limiting theory, bind to the same or
a related (e.g., a structurally similar or spatially proximal)
epitope on human IL-13 as the antibody with which it competes. In a
certain embodiment, the antibody that binds to the same epitope on
human IL-13 as the antibodies of the present invention is a human
monoclonal antibody. Such human monoclonal antibodies can be
prepared and isolated as described in the Examples.
Engineered and Modified Antibodies
[0094] An antibody of the invention further can be prepared using
an antibody having one or more of the V.sub.H and/or V.sub.L
sequences shown herein as starting material to engineer a modified
antibody, which modified antibody may have altered properties from
the starting antibody. An antibody can be engineered by modifying
one or more residues within one or both variable regions (i.e.,
V.sub.H and/or V.sub.L), for example within one or more CDR regions
and/or within one or more framework regions. Additionally or
alternatively, an antibody can be engineered by modifying residues
within the constant region(s), for example to alter the effector
function(s) of the antibody.
[0095] One type of variable region engineering that can be
performed is CDR grafting. Antibodies interact with target antigens
predominantly through amino acid residues that are located in the
six heavy and light chain complementarity determining regions
(CDRs). For this reason, the amino acid sequences within CDRs are
more diverse between individual antibodies than sequences outside
of CDRs. Because CDR sequences are responsible for most
antibody-antigen interactions, it is possible to express
recombinant antibodies that mimic the properties of specific
naturally occurring antibodies by constructing expression vectors
that include CDR sequences from the specific naturally occurring
antibody grafted onto framework sequences from a different antibody
with different properties (see, e.g., Riechmann, L. et al., 1998
Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522-525;
Queen, C. et al., 1989 Proc. Natl. Acad. See. U.S.A.
86:10029-10033; U.S. Pat. No. 5,225,539 to winter, and U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et
al.)
[0096] Accordingly, another embodiment of the invention pertains to
an isolated monoclonal antibody, or antigen binding portion
thereof, comprising a heavy chain variable region comprising CDR1
sequences having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 6-7; CDR2 sequences having an amino acid
sequence of SEQ ID NO: 8; CDR3 sequences having an amino acid
sequence selected from the group consisting of SEQ ID NOs: 9-10,
respectively; and a light chain variable region having CDR1
sequences having an amino acid sequence selected from the group
consisting of SEQ ID NOs: 16-18; CDR2 sequences having an amino
acid sequence of SEQ ID NO: 19; and CDR3 sequences consisting of an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 20-22, respectively. Thus, such antibodies contain the V.sub.H
and V.sub.L CDR sequences of monoclonal antibodies, yet may contain
different framework sequences from these antibodies.
[0097] Such framework sequences can be obtained from public DNA
databases or published references that include germline antibody
gene sequences. For example, germline DNA sequences for human heavy
and light chain variable region genes can be found in the "VBase"
human germline sequence database (available on the Internet at
www.mrc-cpe.cam.ac.uk/vbase), as well as in Kabat, E. A., et al.,
1991 Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242; Tomlinson, I. M., et al., 1992 J. fol.
Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. J Immunol.
24:827-836; the contents of each of which are expressly
incorporated herein by reference.
[0098] An example of framework sequences for use in the antibodies
of the invention are those that are structurally similar to the
framework sequences used by selected antibodies of the invention,
e.g., consensus sequences and/or framework sequences used by
monoclonal antibodies of the invention. The V.sub.H CDR1, 2 and 3
sequences, and the V.sub.L CDR1, 2 and 3 sequences, can be grafted
onto framework regions that have the identical sequence as that
found in the germline immunoglobulin gene from which the framework
sequence derive, or the CDR sequences can be grafted onto framework
regions that contain one or more mutations as compared to the
germline sequences. For example, it has been found that in certain
instances it is beneficial to mutate residues within the framework
regions to maintain or enhance the antigen binding ability of the
antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762
and 6,180,370 to Queen et al).
[0099] Another type of variable region modification is to mutate
amino acid residues within the V.sub.H and/or V.sub.L CDR1, CDR2
and/or CDR3 regions to thereby improve one or more binding
properties (e.g., affinity) of the antibody of interest, known as
"affinity maturation." Site-directed mutagenesis or PCR-mediated
mutagenesis can be performed to introduce the mutation(s) and the
effect on antibody binding, or other functional property of
interest, can be evaluated in in vitro or in vivo assays as
described herein and provided in the Examples. Conservative
modifications (as discussed above) can be introduced. The mutations
may be amino acid substitutions, additions or deletions. Moreover,
typically no more than one, two, three, four or five residues
within a CDR region are altered.
[0100] Accordingly, in another embodiment, the invention provides
isolated anti-IL-13 monoclonal antibodies, or antigen binding
portions thereof, consisting of a heavy chain variable region
having: a V.sub.H CDR1 region consisting of an amino acid sequence
selected from the group having SEQ ID NOs: 6-7 or an amino acid
sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs:
6-7; a V.sub.H CDR2 region having an amino acid sequence of SEQ ID
NO: 8, or an amino acid sequence having one, two, three, four or
five amino acid substitutions, deletions or additions as compared
to SEQ ID NO: 8; a V.sub.H CDR3 region having an amino acid
sequence selected from the group consisting of SEQ ID NOs: 9-10, or
an amino acid sequence having one, two, three, four or five amino
acid substitutions, deletions or additions as compared to SEQ ID
NOs: 9-10; a V.sub.L CDR1 region having an amino acid sequence
selected from the group consisting of SEQ ID NOs: 16-18, or an
amino acid sequence having one, two, three, four or five amino acid
substitutions, deletions or additions as compared to SEQ ID NOs:
16-18; a V.sub.L CDR2 region having an amino acid sequence of SEQ
ID NO: 19, or an amino acid sequence having one, two, three, four
or five amino acid substitutions, deletions or additions as
compared to SEQ ID NO: 19; and a V.sub.L CDR3 region having an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 20-22, or an amino acid sequence having one, two, three, four
or five amino acid substitutions, deletions or additions as
compared to SEQ ID NOs: 20-22.
[0101] Engineered antibodies of the invention include those in
which modifications have been made to framework residues within
V.sub.H and/or V.sub.L, e.g. to improve the properties of the
antibody. Typically such framework modifications are made to
decrease the immunogenicity of the antibody. For example, one
approach is to "backmutate" one or more framework residues to the
corresponding germline sequence. More specifically, an antibody
that has undergone somatic mutation may contain framework residues
that differ from the germline sequence from which the antibody is
derived. Such residues can be identified by comparing the antibody
framework sequences to the germline sequences from which the
antibody is derived. To return the framework region sequences to
their germline configuration, the somatic mutations can be
"backmutated" to the germline sequence by, for example,
site-directed mutagenesis or PCR-mediated mutagenesis. Such
"backmutated" antibodies are also intended to be encompassed by the
invention.
[0102] Another type of framework modification involves mutating one
or more residues within the framework region, or even within one or
more CDR regions, to remove T cell-epitopes to thereby reduce the
potential immunogenicity of the antibody. This approach is also
referred to as "deimmunization" and is described in further detail
in U.S. Patent Publication No. 20030153043 by Carr et al.
[0103] In addition or alternative to modifications made within the
framework or CDR regions, antibodies of the invention may be
engineered to include modifications within the Fc region, typically
to alter one or more functional properties of the antibody, such as
serum half-life, complement fixation, Fc receptor binding, and/or
antigen-dependent cellular cytotoxicity. Furthermore, an antibody
of the invention may be chemically modified (e.g., one or more
chemical moieties can be attached to the antibody) or be modified
to alter its glycosylation, again to alter one or more functional
properties of the antibody. Each of these embodiments is described
in further detail below. The numbering of residues in the Fc region
is that of the EU index of Kabat.
[0104] In one embodiment, the hinge region of CH1 is modified such
that the number of cysteine residues in the hinge region is
altered, e.g., increased or decreased. This approach is described
further in U.S. Pat. No. 5,677,425 by Bodmer et al. The number of
cysteine residues in the hinge region of CH1 is altered to, for
example, facilitate assembly of the light and heavy chains or to
increase or decrease the stability of the antibody.
[0105] In another embodiment, the Fc hinge region of an antibody is
mutated to decrease the biological half-life of the antibody. More
specifically, one or more amino acid mutations are introduced into
the CH2-CH3 domain interface region of the Fc-hinge fragment such
that the antibody has impaired Staphylococcyl protein A (SpA)
binding relative to native Fc-hinge domain SpA binding. This
approach is described in further detail in U.S. Pat. No. 6,165,745
by Ward et al.
[0106] In another embodiment, the antibody is modified to increase
its biological half-life. Various approaches are possible. For
example, one or more of the following mutations can be introduced:
T252L, T254S, T256F, as described in U.S. Pat. No. 6,277,375 to
Ward. Alternatively, to increase the biological half life, the
antibody can be altered within the CH1 or CL region to contain a
salvage receptor binding epitope taken from two loops of a CH2
domain of an Fc region of an IgG, as described in U.S. Pat. Nos.
5,869,046 and 6,121,022 by Presta et al.
[0107] In yet other embodiments, the Fc region is altered by
replacing at least one amino acid residue with a different amino
acid residue to alter the effector functions of the antibody. For
example, one or more amino acids can be replaced with a different
amino acid residue such that the antibody has an altered affinity
for an effector ligand but retains the antigen-binding ability of
the parent antibody. The effector ligand to which affinity is
altered can be, for example, an Fc receptor or the C1 component of
complement. This approach is described in further detail in U.S.
Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
[0108] In another embodiment, one or more amino acids selected from
amino acid residues can be replaced with a different amino acid
residue such that the antibody has altered C1q binding and/or
reduced or abolished complement dependent cytotoxicity (CDC). This
approach is described in further detail in U.S. Pat. No. 6,194,551
by Idusogie et at.
[0109] In another embodiment, one or more amino acid residues are
altered to thereby alter the ability of the antibody to fix
complement. This approach is described further in PCT Publication
WO 94/29351 by Bodmer et al.
[0110] In yet another embodiment, the Fc region is modified to
increase the ability of the antibody to mediate antibody dependent
cellular cytotoxicity (ADCC) and/or to increase the affinity of the
antibody for an Fc.gamma. receptor by modifying one or more amino
acids. This approach is described further in PCT Publication WO
00/42072 by Presta. Moreover, the binding sites on human IgG1 for
Fc.gamma.RI, Fc.gamma.RII, Fc.gamma.RIII and FcRn have been mapped
and variants with improved binding have been described (see
Shields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604).
[0111] In still another embodiment, the glycosylation of an
antibody is modified. For example, an aglycoslated antibody can be
made (i.e., the antibody lacks glycosylation). Glycosylation can be
altered to, for example, increase the affinity of the antibody for
"antigen`. Such carbohydrate modifications can be accomplished by;
for example, altering one or more sites of glycosylation within the
antibody sequence. For example, one or more amino acid
substitutions can be made that result in elimination of one or more
variable region framework glycosylation sites to thereby eliminate
glycosylation at that site. Such aglycosylation may increase the
affinity of the antibody for antigen. Such an approach is described
in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 by Co
et al.
[0112] Additionally or alternatively, an antibody can be made that
has an altered type of glycosylation, such as a hypofucosylated
antibody having reduced amounts of fucosyl residues or an antibody
having increased bisecting GlcNac structures. Such altered
glycosylation patterns have been demonstrated to increase the ADCC
ability of antibodies. Such carbohydrate modifications can be
accomplished by, for example, expressing the antibody in a host
cell with altered glycosylation machinery. Cells with altered
glycosylation machinery have been described in the art and can be
used as host cells in which to express recombinant antibodies of
the invention to thereby produce an antibody with altered
glycosylation. For example, EP 1,176,195 by Hang et al. describes a
cell line with a functionally disrupted FUT8 gene, which encodes a
fucosyl transferase, such that antibodies expressed in such a cell
line exhibit hypofucosylation. PCT Publication WO 03/035835 by
Presta describes a variant CHO cell line, Lec13 cells, with reduced
ability to attach fucose to Asn(297)-linked carbohydrates, also
resulting in hypofucosylation of antibodies expressed in that host
cell (see also Shields, R. L. et al., 2002 J. Biol. Chem.
277:26733-26740). PCT Publication WO 99/54342 by Umana et al.
describes cell lines engineered to express glycoprotein-modifying
glycosyl transferases (e.g., beta(1,4)-N
acetylglucosaminyltransferase III (GnTIII)) such that antibodies
expressed in the engineered cell lines exhibit increased bisecting
GlcNac structures which results in increased ADCC activity of the
antibodies (see also Umana et al., 1999 Nat. Biotech.
17:176-180).
[0113] Another modification of the antibodies herein that is
contemplated by the invention is pegylation. An antibody can be
pegylated to, for example, increase the biological (e.g., serum)
half-life of the antibody. To pegylate an antibody, the antibody,
or fragment thereof, typically is reacted with polyethylene glycol
(PEG), such as a reactive ester or aldehyde derivative of PEG,
under conditions in which one or more PEG groups become attached to
the antibody or antibody fragment. The pegylation can be carried
out by an acylation reaction or an alkylation reaction with a
reactive PEG molecule (or an analogous reactive water-soluble
polymer). As used herein, the term "polyethylene glycol" is
intended to encompass any of the forms of PEG that have been used
to derivatize other proteins, such as mono (C1-C10) alkoxy- or
aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In
certain embodiments, the antibody to be pegylated is an
aglycosylated antibody. Methods for pegylating proteins are known
in the art and can be applied to the antibodies of the invention.
See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384
by Ishikawa et al.
Methods of Engineering Antibodies
[0114] As discussed above, the anti-IL-13 antibodies having V.sub.H
and V.sub.L sequences shown herein can be used to create new
anti-IL-13 antibodies by modifying the V.sub.H and/or V.sub.L
sequences, or the constant region(s) attached thereto. Thus, in
another aspect of the invention, the structural features of an
anti-IL-13 antibody of the invention are used to create
structurally related anti-IL-13 antibodies that retain at least one
functional property of the antibodies of the invention, such as
binding to human IL-13 and also inhibiting one or more functional
properties of IL-13 (e.g., receptor binding, inhibition of mediator
release).
[0115] For example, one or more CDR regions of the antibodies of
the present invention, or mutations thereof, can be combined
recombinantly with known framework regions and/or other CDRs to
create additional, recombinantly-engineered, anti-IL-13 antibodies
of the invention, as discussed above. Other types of modifications
include those described in the previous section. The starting
material for the engineering method is one or more of the V.sub.H
and/or V.sub.L sequences provided herein, or one or more CDR
regions thereof. To create the engineered antibody, it is not
necessary to actually prepare (i.e., express as a protein) an
antibody having one or more of the V.sub.H and/or V.sub.L sequences
provided herein, or one or more CDR regions thereof. Rather, the
information contained in the sequence(s) is used as the starting
material to create a "second generation" sequence(s) derived from
the original sequence(s) and then the "second generation"
sequence(s) is prepared and expressed as a protein.
[0116] Accordingly, in another embodiment, the invention provides a
method for preparing an anti-IL-13 antibody consisting of: a heavy
chain variable region antibody sequence having a CDR1 sequence
selected from the group consisting of SEQ ID NOs: 6-7, a CDR2
sequence of SEQ ID NO: 8 and/or a CDR3 sequence selected from the
group consisting of SEQ ID NOs: 9-10; and a light chain variable
region antibody sequence having a CDR1 sequence selected from the
group consisting of SEQ ID NOs: 16-18, a CDR2 sequence of SEQ ID
NO: 19 and/or a CDR3 sequence selected from the group consisting of
SEQ ID NOs: 20-22; altering at least one amino acid residue within
the heavy chain variable region antibody sequence and/or the light
chain variable region antibody sequence to create at least one
altered antibody sequence; and expressing the altered antibody
sequence as a protein.
[0117] Standard molecular biology techniques can be used to prepare
and express the altered antibody sequence. The antibody encoded by
the altered antibody sequence(s) is one that retains one, some or
all of the functional properties of the anti-IL-13 antibodies
described herein, which functional properties include, but are not
limited to, specifically binding to human IL-13; and the antibody
exhibits at least one of the following functional properties: the
antibody inhibits binding of IL-13 protein to the IL-13 receptor,
or the antibody inhibits IL-13 receptor binding preventing or
ameliorating an inflammatory, fibrotic or allergic condition,
particularly an inflammatory or obstructive airways disease, or the
antibody inhibits IL-13 receptor binding thereby preventing or
ameliorating asthma.
[0118] The altered antibody may exhibit one or more, two or more,
or three or more of the functional properties discussed above.
[0119] The functional properties of the altered antibodies can be
assessed using standard assays available in the art and/or
described herein, such as those set forth in the Examples (e.g.,
ELISAs).
[0120] In certain embodiments of the methods of engineering
antibodies of the invention, mutations can be introduced randomly
or selectively along all or part of an anti-IL-13 antibody coding
sequence and the resulting modified anti-IL-13 antibodies can be
screened for binding activity and/or other functional properties as
described herein. Mutational methods have been described in the
art. For example, PCT Publication WO 02/092780 by Short describes
methods for creating and screening antibody mutations using
saturation mutagenesis, synthetic ligation assembly, or a
combination thereof. Alternatively, PCT Publication WO 03/074679 by
Lazar et al. describes methods of using computational screening
methods to optimize physiochemical properties of antibodies.
Nucleic Acid Molecules Encoding Antibodies of the Invention
[0121] Another aspect of the invention pertains to nucleic acid
molecules that encode the antibodies of the invention. The nucleic
acids may be present in whole cells, in a cell lysate, or may be
nucleic acids in a partially purified or substantially pure form. A
nucleic acid is "isolated" or "rendered substantially pure" when
purified away from other cellular components or other contaminants,
e.g., other cellular nucleic acids or proteins, by standard
techniques, including alkaline/SDS treatment, CsCl banding, column
chromatography, agarose gel electrophoresis and others well known
in the art. See, F. Ausubel, et al., ed. 1987 Current Protocols in
Molecular Biology, Greene Publishing and Wiley Interscience, New
York. A nucleic acid of the invention can be, for example, DNA or
RNA and may or may not contain intronic sequences. In an
embodiment, the nucleic acid is a cDNA molecule. The nucleic acid
may be present in a vector such as a phage display vector, or in a
recombinant plasmid vector.
[0122] Nucleic acids of the invention can be obtained using
standard molecular biology techniques. For antibodies expressed by
hybridomas (e.g., hybridomas prepared from transgenic mice carrying
human immunoglobulin genes as described further below), cDNAs
encoding the light and heavy chains of the antibody made by the
hybridoma can be obtained by standard PCR amplification or cDNA
cloning techniques. For antibodies obtained from an immunoglobulin
gene library (e.g., using phage display techniques), nucleic acid
encoding the antibody can be recovered from various phage clones
that are members of the library.
[0123] Once DNA fragments encoding V.sub.H and V.sub.L segments are
obtained, these DNA fragments can be further manipulated by
standard recombinant DNA techniques, for example to convert the
variable region genes to full-length antibody chain genes, to Fab
fragment genes or to an scFv gene. In these manipulations, a
V.sub.L- or V.sub.H-encoding DNA fragment is operatively linked to
another DNA molecule, or to a fragment encoding another protein,
such as an antibody constant region or a flexible linker. The term
"operatively linked", as used in this context, is intended to mean
that the two DNA fragments are joined in a functional manner, for
example, such that the amino acid sequences encoded by the two DNA
fragments remain in-frame, or such that the protein is expressed
under control of a desired promoter.
[0124] The isolated DNA encoding the V.sub.H region can be
converted to a full-length heavy chain gene by operatively linking
the V.sub.H-encoding DNA to another DNA molecule encoding heavy
chain constant regions (CH1, CH2 and CH3). The sequences of human
heavy chain constant region genes are known in the art (see e.g.,
Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The heavy chain constant region can be an IgG1,
IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region. For a Fab
fragment heavy chain gene, the V.sub.H-encoding DNA can be
operatively linked to another DNA molecule encoding only the heavy
chain CH1 constant region.
[0125] The isolated DNA encoding the V.sub.L region can be
converted to a full-length light chain gene (as well as to a Fab
light chain gene) by operatively linking the V.sub.L-encoding DNA
to another DNA molecule encoding the light chain constant region,
CL. The sequences of human light chain constant region genes are
known in the art (see e.g., Kabat, E. A., et al., 1991 Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242) and DNA
fragments encompassing these regions can be obtained by standard
PCR amplification. The light chain constant region can be a kappa
or a lambda constant region.
[0126] To create an scFv gene, the V.sub.H- and V.sub.L-encoding
DNA fragments are operatively linked to another fragment encoding a
flexible linker, e.g., encoding the amino acid sequence
(Gly4-Ser).sub.3, such that the V.sub.H and V.sub.L sequences can
be expressed as a contiguous single-chain protein, with the V.sub.L
and V.sub.H regions joined by the flexible linker (see e.g., Bird
et al., 1988 Science 242:423-426; Huston et at., 1988 Proc. Natl.
Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990 Nature
348:552-554).
Production of Monoclonal Antibodies of the Invention
[0127] Monoclonal antibodies (mAbs) can be produced by a variety of
techniques, including conventional monoclonal antibody methodology
e.g., the standard somatic cell hybridization technique of Kohler
and Milstein, 1975 Nature 256: 495. Many techniques for producing
monoclonal antibody can be employed e.g., viral or oncogenic
transformation of B lymphocytes.
[0128] An animal system for preparing hybridomas is the murine
system. Hybridoma production in the mouse is a well established
procedure. Immunization protocols and techniques for isolation of
immunized splenocytes for fusion are known in the art. Fusion
partners (e.g., murine myeloma cells) and fusion procedures are
also known.
[0129] Chimeric or humanized antibodies of the present invention
can be prepared based on the sequence of a murine monoclonal
antibody prepared as described above. DNA encoding the heavy and
light chain immunoglobulins can be obtained from the murine
hybridoma of interest and engineered to contain non-murine (e.g.,
human) immunoglobulin sequences using standard molecular biology
techniques. For example, to create a chimeric antibody, the murine
variable regions can be linked to human constant regions using
methods known in the art (see e.g., U.S. Pat. No. 4,816,567 to
Cabilly et al.). To create a humanized antibody, the murine CDR
regions can be inserted into a human framework using methods known
in the art (see e.g., U.S. Pat. No. 5,225,539 to Winter, and U.S.
Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and, 6,180,370 to. Queen.
et al.
[0130] In a certain embodiment, the antibodies of the invention are
human monoclonal antibodies. Such human monoclonal antibodies
directed against IL-13 can be generated using transgenic or
transchromosomic mice carrying parts of the human immune system
rather than the mouse system. These transgenic and transchromosomic
mice include mice referred to herein as HuMAb mice and KM mice,
respectively, and are collectively referred to herein as "human Ig
mice."
[0131] The HuMAb mouses (Medarex, Inc.) contains human
immunoglobulin gene miniloci that encode un-rearranged human heavy
(.mu. and .gamma.) and .kappa. light chain immunoglobulin
sequences, together with targeted mutations that inactivate the
endogenous .mu. and .kappa. chain loci (see e.g., Lonberg, et al.,
1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit
reduced expression of mouse IgM or .kappa., and in response to
immunization, the introduced human heavy and light chain transgenes
undergo class switching and somatic mutation to generate high
affinity human IgG.kappa. monoclonal (Lonberg, N. et al., 1994
supra; reviewed in Lonberg, N., 1994 Handbook of Experimental
Pharmacology 113:49-101; Lonberg, N. and Huszar, D., 1995 Intern.
Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N., 1995 Ann.
N.Y. Acad. Sci. 764:536-546). The preparation and use of HuMAb
mice, and the genomic modifications carried by such mice, is
further described in Taylor, L. et al., 1992 Nucleic Acids Research
20:6287-6295; Chen, J. et at., 1993 International Immunology 5:
647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA
94:3720-3724; Choi et al., 1993 Nature Genetics 4:117-123; Chen, J.
et al., 1993 EMBO J. 12: 821-830; Tuaillon et al., 1994 J. Immunol.
152:2912-2920; Taylor, L. et al., 1994 International Immunology
579-591; and Fishwild, D. et al., 1996 Nature Biotechnology 14:
845-851, the contents of all of which are hereby specifically
incorporated by reference in their entirety. See further, U.S. Pat.
Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650;
5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; all to
Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCT
Publication Nos. WO 92103918, WO 93/12227, WO 94/25585, WO
97113852, WO 98/24884 and WO 99/45962, all to Lonberg and Kay; and
PCT Publication No. WO 01/14424 to Korman et al.
[0132] In another embodiment, human antibodies of the invention can
be raised using a mouse that carries human immunoglobulin sequences
on transgenes and transchomosomes such as a mouse that carries a
human heavy chain transgene and a human light chain
transchromosome. Such mice, referred to herein as "KM mice", are
described in detail in PCT Publication WO 02/43478 to Ishida et
al.
[0133] Still further, alternative transgenic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-IL-13 antibodies of the invention. For
example, an alternative transgenic system referred to as the
Xenomouse (Abgenix, Inc.) can be used; such mice are described in,
for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598;
6,150,584 and 6,162,963 to Kucherlapati et al.
[0134] Moreover, alternative transchromosomic animal systems
expressing human immunoglobulin genes are available in the art and
can be used to raise anti-IL-13 antibodies of the invention. For
example, mice carrying both a human heavy chain transchromosome and
a human light chain tranchromosome, referred to as "TC mice" can be
used; such mice are described in Tomizuka et al., 2000 Proc. Natl.
Acad. Sci. USA 97:722-727. Furthermore, cows carrying human heavy
and light chain transchromosomes have been described in the art
(Kuroiwa et al., 2002 Nature Biotechnology 20:889-894) and can be
used to raise anti-IL-13 antibodies of the invention.
[0135] Human monoclonal antibodies of the invention can also be
prepared using phage display methods for screening libraries of
human immunoglobulin genes. Such phage display methods for
isolating human antibodies are established in the art. See for
example: U.S. Pat. Nos. 5,223,409; 5,403,484; and 5,571,698 to
Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 to Dower et
al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty et al.;
and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;
6,582,915 and 6,593,081 to Griffiths et al.
[0136] Human monoclonal antibodies of the invention can also be
prepared using SCID mice into which human immune cells have been
reconstituted such that a human antibody response can be generated
upon immunization. Such mice are described in, for example, U.S.
Pat. Nos. 5,476,996 and 5,698,767 to Wilson et al.
Generation of Human Monoclonal Antibodies Against IL-13
[0137] Purified recombinant human (hr) IL-13 conjugated to Pan DR T
helper Epitopes (PADRE), is used as the antigen. Fully human
monoclonal antibodies to IL-13 are prepared using HCo7 strains of
HuMab transgenic mice which express human antibody genes. In this
mouse strain, the endogenous mouse kappa light chain gene can be
homozygously disrupted as described in Chen et al., 1993 EMBO J.
12:811-820 and the endogenous mouse heavy chain gene can be
homozygously disrupted as described in Example 1 of PCT Publication
WO 01109187. This mouse strain carries a human kappa light chain
transgene, KCoS, as described in Fishwild et al., 1996 Nature
Biotechnology 14:845-851 and the HCo7 human heavy chain transgene
as described in U.S. Pat. Nos. 5,545,806; 5,625,825; and
5,545,807.
[0138] To generate fully human monoclonal antibodies to IL-13 of
the invention, HuMab mice are immunized with a mixture of purified
recombinant IL-13 derived from HEK-EBNA/PADRE conjugate (42
ug/mouse) and Quil A (15 ug/mouse, Accurate Chemical). General
immunization schemes for HuMab mice are described in Lonberg, N. et
al., 1994 Nature 368(6474): 856-859; Fishwild, D. et al., 1996
Nature Biotechnology 14:845-851 and PCT Publication WO 98/24884.
Transgenic mice are immunized either intravenously (IV), or
subcutaneously (SC) between day 1-71. Mice are boosted
intravenously with antigen (without adjuvant) 2 days before
sacrifice and removal of the spleen. RNA was isolated from spleens
using the Nucleospin RNA II isolation kit (BD
Biosciences/Clontech). The RNA was used to generate a phage display
library of randomly assorted H and L chain variable domains in a
Fab phage display vector as described in U.S. Pat. No. 6,794,132.
The phage display library was subjected to five rounds of selection
using biotinylated hrIL-13 in a solution-phase equilibrium binding
protocol as described in the patent. The first four rounds of
selection employed hrIL-13 at 10.sup.-8 M and the last round of
selection employed hrIL-13 at 10.sup.-9 M. The final signal to
noise ratio determined by counting pfu's recovered in the presence
of antigen divided by pfu's recovered in the absence of antigen was
37 for this library, indicating that greater than 90% of the phage
selected were expressing antibodies that bound hrIL-13. The phage
display library was then subcloned into a plasmid vector for the
expression of soluble Fab as described in U.S. Pat. No.
6,794,132.
Generation of Transfectomas Producing Monoclonal Antibodies
[0139] Antibodies of the invention also can be produced in a host
cell transfectoma using, for example, a combination of recombinant
DNA techniques and gene transfection methods as is well known in
the art (e.g., Morrison, S. (1985) Science 229:1202).
[0140] For example, to express the antibodies, or antibody
fragments thereof, DNAs encoding partial or full-length light and
heavy chains, can be obtained by standard molecular biology
techniques (e.g., PCR amplification or cDNA cloning using a
hybridoma that expresses the antibody of interest) and the DNAs can
be inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy chain gene can be inserted into separate
vector or, more typically, both genes are inserted into the same
expression vector. The antibody genes are inserted into the
expression vector by standard methods (e.g., ligation of
complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are present).
The light and heavy chain variable regions of the antibodies
described herein can be used to create full-length antibody genes
of any antibody isotype by inserting them into expression vectors
already encoding heavy chain constant and light chain constant
regions of the desired isotype such that the V.sub.H segment is
operatively linked to the CH segment(s) within the vector and the
V.sub.L segment is operatively linked to the CL segment within the
vector. Additionally or alternatively, the recombinant expression
vector can encode a signal peptide that facilitates secretion of
the antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked in
frame to the amino terminus of the antibody chain gene. The signal
peptide can be an immunoglobulin signal peptide or a heterologous
signal peptide (i.e., a signal peptide from a non-immunoglobulin
protein).
[0141] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel (Gene Expression Technology.
Methods in Enzymology 185, Academic Press, San Diego, Calif. 1990).
It will be appreciated by those skilled in the art that the design
of the expression vector, including the selection of regulatory
sequences, may depend on such factors as the choice of the host
cell to be transformed, the level of expression of protein desired,
etc. Regulatory sequences for mammalian host cell expression
include viral elements that direct high levels of protein
expression in mammalian cells, such as promoters and/or enhancers
derived from cytomegalovirus (CMV), Simian Virus 40 (SV40),
adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and
polyoma. Alternatively, nonviral regulatory sequences may be used,
such as the ubiquitin promoter or P-globin promoter. Still further,
regulatory elements composed of sequences from different sources,
such as the SRa promoter system, which contains sequences from the
SV40 early promoter and the long terminal repeat of human T cell
leukemia virus type 1 (Takebe, Y. et al., 1988 Mol. Cell. Biol.
8:466-472).
[0142] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells
with methotrexate selection/amplification) and the neo gene (for
G418 selection).
[0143] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. It is theoretically possible to express the antibodies of the
invention in either prokaryotic or eukaryotic host cells.
Expression of antibodies in eukaryotic cells, in particular
mammalian host cells, is discussed because such eukaryotic cells,
and in particular mammalian cells, are more likely than prokaryotic
cells to assemble and secrete a properly folded and immunologically
active antibody. Prokaryotic expression of antibody genes has been
reported to be ineffective for production of high yields of active
antibody (Boss, M. A. and Wood, C. R., 1985 Immunology Today
6:12-13).
[0144] Mammalian host cells for expressing the recombinant
antibodies of the invention include Chinese Hamster Ovary (CHO
cells) (including dhfr-CHO cells, described Urlaub and Chasin, 1980
Proc. Natl. Acad. Sci. USA 77:4216-4220 used with a DH FR
selectable marker, e.g., as described in R. J. Kaufman and P. A.
Sharp, 1982 Mol. Biol. 159:601-621, NSO myeloma cells, COS cells
and SP2 cells. When recombinant expression vectors encoding
antibody genes are introduced into mammalian host cells, the
antibodies are produced by culturing the host cells for a period of
time sufficient to allow for expression of the antibody in the host
cells or secretion of the antibody into the culture medium in which
the host cells are grown. Antibodies can be recovered from the
culture medium using standard protein purification methods.
Immunoconjugates
[0145] In another aspect, the present invention features an
anti-IL-13 antibody, or a fragment thereof, conjugated to a
therapeutic moiety, such as a cytotoxin, a drug (e.g., an
immunosuppressant) or a radiotoxin. Such conjugates are referred to
herein as "immunoconjugates". Immunoconjugates that include one or
more cytotoxins are referred to as "immunotoxins." A cytotoxin or
cytotoxic agent includes any agent that is detrimental to (e.g.,
kills) cells. Examples include taxon, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, t. colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents also include, for example,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), ablating
agents (e.g., mechlorethamine, thioepa chloraxnbucil, meiphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin, anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine).
[0146] Other examples of therapeutic cytotoxins that can be
conjugated to an antibody of the invention include duocarmycins,
calicheamicins, maytansines and auristatins, and derivatives
thereof. An example of a calicheamicin antibody conjugate is
commercially available (Mylotarg.TM.; Wyeth-Ayerst).
[0147] Cytotoxins can be conjugated to antibodies of the invention
using linker technology available in the art. Examples of linker
types that have been used to conjugate a cytotoxin to an antibody
include, but are not limited to, hydrazones, thioethers, esters,
disulfides and peptide-containing linkers. A linker can be chosen
that is, for example, susceptible to cleavage by low pH within the
lysosomal compartment or susceptible to cleavage by proteases, such
as proteases preferentially expressed in tumor tissue such as
cathepsins (e.g., cathepsins B, C, D).
[0148] For further discussion of types of cytotoxins, linkers and
methods for conjugating therapeutic agents to antibodies, see also
Saito, G. et al., 2003 Adv. Drug Deliv. Rev. 55:199-215; Trail, P.
A. et al., 2003 Cancer Immunol. Immunother. 52:328-337; Payne, G.,
2003 Cancer Cell 3:207-212; Allen, T. M., 2002 Nat. Rev. Cancer
2:750-763; Pastan, I. and Kreitman, R. J., 2002 Curr. Opin.
Investig. Drugs 3:1089-1091; Senter, P. D. and Springer, C. J.,
2001 Adv. Drug Deliv. Rev. 53:247-264.
[0149] Antibodies of the present invention also can be conjugated
to a radioactive isotope to generate cytotoxic
radiopharmaceuticals, also referred to as radioimmunoconjugates.
Examples of radioactive isotopes that can be conjugated to
antibodies for use diagnostically or therapeutically include, but
are not limited to, iodine.sup.131, indium.sup.111, yttrium.sup.90,
and lutetium.sup.177. Method for preparing radioimmunconjugates are
established in the art. Examples of radioimmunoconjugates are
commercially available, including Zevalin.TM. (DEC Pharmaceuticals)
and Bexxar.TM. (Corixa Pharmaceuticals), and similar methods can be
used to prepare radioimmunoconjugates using the antibodies of the
invention.
[0150] The antibody conjugates of the invention can be used to
modify a given biological response, and the drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, an enzymatically active toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor or
interferon-.gamma.; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other growth factors.
[0151] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Amon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et at., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
Bispecific Molecules
[0152] In another aspect, the present invention features bispecific
molecules comprising an anti-IL-13 antibody, or a fragment thereof,
of the invention. An antibody of the invention, or antigen-binding
portions thereof, can be derivatized or linked to another
functional molecule, e.g., another peptide or protein (e.g.,
another antibody or ligand for a receptor) to generate a bispecific
molecule that binds to at least two different binding sites or
target molecules. The antibody of the invention may in fact be
derivatized or linked to more than one other functional molecule to
generate multi-specific molecules that bind to more than two
different binding sites and/or target molecules; such
multi-specific molecules are also intended to be encompassed by the
term "bispecific molecule" as used herein. To create a bispecific
molecule of the invention, an antibody of the invention can be
functionally linked (e.g., by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other binding
molecules, such as another antibody, antibody fragment, peptide or
binding mimetic, such that a bispecific molecule results.
[0153] Accordingly, the present invention includes bispecific
molecules comprising at least one first binding specificity for
IL-13 and a second binding specificity for a second target epitope.
For example, the second target epitope is an Fc receptor, e.g.,
human Fc.gamma.RI (CD64) or a human Fc.alpha. receptor (CD89).
Therefore, the invention includes bispecific molecules capable of
binding both to Fc.gamma.R, Fc.alpha.R or Fc.epsilon.R expressing
effector cells (e.g., monocytes, macrophages or polymorphonuclear
cells (PMNs), and to target cells expressing IL-13. These
bispecific molecules target IL-13 expressing cells to effector cell
and trigger Fc receptor-mediated effector cell activities, such as
phagocytosis of an IL-13 expressing cells, antibody dependent
cell-mediated cytotoxicity (ADCC), cytokine release, or generation
of superoxide anion.
[0154] Additionally, for the invention in which the bispecific
molecule is multi-specific, the molecule can further include a
third binding specificity, in addition to an anti-Fc binding
specificity and an anti-IL-13 binding specificity. For example, the
third binding specificity could be an anti-enhancement factor (EF)
portion, e.g., a molecule which binds to a surface protein involved
in cytotoxic activity and thereby increases the immune response
against the target cell. The "anti-enhancement factor portion"
could be an antibody, functional antibody fragment or a ligand that
binds to a given molecule, e.g., an antigen or a receptor, and
thereby results in an enhancement of the effect of the binding
determinants for the Fc receptor or target cell antigen.
[0155] The "anti-enhancement factor portion" can bind an Fc
receptor or a target cell antigen. Alternatively, the
anti-enhancement factor portion could bind to an entity that is
different from the entity to which the first and second binding
specificities bind. For example, the anti-enhancement factor
portion can bind a cytotoxic T-cell (e.g. by CD2, CD3, CD8, CD28,
CD4, CD44, ICAM-1 or other immune cell that results in an increased
immune response against the target cell).
[0156] In one embodiment, the bispecific molecules of the invention
comprise as a binding specificity at least one antibody, or an
antibody fragment thereof, including, e.g., an Fab, Fab',
F(ab').sub.2, Fv, or a single chain Fv. The antibody may also be a
light chain or heavy chain dimer, or any minimal fragment thereof
such as a Fv or a single chain construct as described in Ladner et
al. U.S. Pat. No. 4,946,778, the contents of which is expressly
incorporated by reference.
[0157] In one embodiment, the binding specificity for an Fc.gamma.
receptor is provided by a monoclonal antibody, the binding of which
is not blocked by human immunoglobulin G (IgG). As used herein, the
term "IgG receptor" refers to any of the eight .gamma.-chain genes
located on chromosome 1. These genes encode a total of twelve
transmembrane or soluble receptor isoforms which are grouped into
three Fy receptor classes: Fc.gamma.RI (CD64), Fc.gamma.RII (CD32),
and Fc.gamma.RIII (CD 16). In another embodiment, the Fc.gamma.
receptor is a human high affinity Fc.gamma.RI. The human
Fc.gamma.RI is a 72 kDa molecule, which shows high affinity for
monomeric IgG (10.sup.8-10.sup.9 M.sup.-1).
[0158] The production and characterization of certain
anti-Fc.gamma. monoclonal antibodies are described by Fanger et at.
in PCT Publication WO 88/00052 and in U.S. Pat. No. 4,954,617, the
teachings of which are fully incorporated by reference herein.
These antibodies bind to an epitope of Fc.gamma.RI, Fc.gamma.RII or
Fc.gamma.RIII at a site which is distinct from the Fc.gamma.
binding site of the receptor and, thus, their binding is not
blocked substantially by physiological levels of IgG. Specific
anti-Fc.gamma.RI antibodies useful in this invention are mAb 22,
mAb 32, mAb 44, mAb 62 and mAb 197. The hybridoma producing mAb 32
is available from the American Type Culture Collection, ATCC
Accession No. HB9469. In other embodiments, the anti-Fc.gamma.
receptor antibody is a humanized form of monoclonal antibody 22
(H22). The production and characterization of the H22 antibody is
described in Graziano, R. F. et al., 1995 J. Immunol 155 (10):
4996-5002 and PCT Publication WO 94/10332. The 1122 antibody
producing cell line was deposited at the American Type Culture
Collection under the designation HA022CL1 and has the accession no.
CRL 11177.
[0159] In still other embodiments, the binding specificity for an
Fc receptor is provided by an antibody that binds to a human IgA
receptor, e.g., an Fc-alpha receptor (Fc.alpha.RI (CD89), the
binding of which does not have to be blocked by human
immunoglobulin A (IgA). The term "IgA receptor" is intended to
include the gene product of one a gene (Fc.alpha.RI) located on
chromosome 19. This gene is known to encode several alternatively
spliced transmembrane isoforms of 55 to 110 kDa. Fc.alpha.RI (CD89)
is constitutively expressed on monocytes/macrophages, eosinophilic
and neutrophilic granulocytes, but not on non-effector cell
populations. Fc.alpha.RI has medium affinity (5.times.10.sup.7
M.sup.-1) for both IgA1 and IgA2, which is increased upon exposure
to cytokines such as G-CSF or GM-CSF (Morton, H. C. et al., 1996
Critical Reviews in Immunology 116:423-440). Four
Fc.alpha.RI-specific monoclonal antibodies, identified as A3, A59,
A62 and A77, which bind Fc.alpha.RI outside the IgA ligand binding
domain, have been described (Monteiro, R. C. et al., 1992 J.
Immunol. 148:1764).
[0160] Fc.alpha.RI and Fc.gamma.RI are trigger receptors for use in
the bispecific molecules of the invention because they are
expressed primarily on immune effector cells, e.g., monocytes,
PMNs, macrophages and dendritic cells; expressed at high levels
(e.g., 5,000-100,000 per cell); mediators of cytotoxic activities
(e.g., ADCC, phagocytosis); mediate enhanced antigen presentation
of antigens, including self-antigens, targeted to them.
[0161] Other antibodies which can be employed in the bispecific
molecules of the invention are murine, chimeric and humanized
monoclonal antibodies.
[0162] The bispecific molecules of the present invention can be
prepared by conjugating the constituent binding specificities,
e.g., the anti-FcR and anti-IL-13 binding specificities, using
methods known in the art. For example, each binding specificity of
the bispecific molecule can be generated separately and then
conjugated to one another. When the binding specificities are
proteins or peptides, a variety of coupling or cross-linking agents
can be used for covalent conjugation. Examples of cross-linking
agents include protein A, carbodiimide,
N-succinimidyl-5-acetyl-thioacetate (SATA),
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide
(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and
sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate
(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med.
160:1686; Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA
82:8648). Other methods include those described in Paulus, 1985
Behring Ins. Mitt. No. 78, 118-132; Brennan et al., 1985 Science
229:81-83), and Glennie et al., 1987 J. Immunol. 139: 2367-2375).
Conjugating agents are SATA and sulfo-SMCC, both available from
Pierce Chemical Co. (Rockford, Ill.).
[0163] When the binding specificities are antibodies, they can be
conjugated by sulfhydryl bonding of the C-terminus hinge regions of
the two heavy chains. In a particularly embodiment, the hinge
region is modified to contain an odd number of sulfhydryl residues,
for example one, prior to conjugation.
[0164] Alternatively, both binding specificities can be encoded in
the same vector and expressed and assembled in the same host cell.
This method is particularly useful where the bispecific molecule is
a mAb.times.mAb, mAb.times.Fab, Fab.times.F(ab').sub.2 or
ligand.times.Fab fusion protein. A bispecific molecule of the
invention can be a single chain molecule comprising one single
chain antibody and a binding determinant, or a single chain
bispecific molecule comprising two binding determinants. Bispecific
molecules may comprise at least two single chain molecules. Methods
for preparing bispecific molecules are described for example in
U.S. Pat. No. 5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No.
4,881,175; U.S. Pat. No. 5,132,405; U.S. Pat. No. 5,091,513; U.S.
Pat. No. 5,476,786; U.S. Pat. No. 5,013,653; U.S. Pat. No.
5,258,498; and U.S. Pat. No. 5,482,858.
[0165] Binding of the bispecific molecules to their specific
targets can be confirmed by, for example, enzyme-linked
immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis,
bioassay (e.g., growth inhibition), or Western Blot assay. Each of
these assays generally detects the presence of protein-antibody
complexes of particular interest by employing a labeled reagent
(e.g., an antibody) specific for the complex of interest. For
example, the FcR-antibody complexes can be detected using e.g., an
enzyme-linked antibody or antibody fragment which recognizes and
specifically binds to the antibody-FcR complexes. Alternatively,
the complexes can be detected using any of a variety of other
immunoassays. For example, the antibody can be radioactively 4
labeled and used in a radioimmunoassay (RIA) (see, for example,
Weintraub; B., Principles of Radioimmunoassays, Seventh Training
Course on Radioligand Assay Techniques, The Endocrine Society,
March, 1986, which is incorporated by reference herein). The
radioactive isotope can be detected by such means as the use of a
.gamma. counter or a scintillation counter or by
autoradiography.
Pharmaceutical Compositions
[0166] In another aspect, the present invention provides a
composition, e.g., a pharmaceutical composition, containing one or
a combination of monoclonal antibodies, or antigen-binding
portion(s) thereof, of the present invention, formulated together
with a pharmaceutically acceptable carrier. Such compositions may
include one or a combination of (e.g., two or more different)
antibodies, or immunoconjugates or bispecific molecules of the
invention. For example, a pharmaceutical composition of the
invention can comprise a combination of antibodies (or
immunoconjugates or bispecifics) that bind to different epitopes on
the target antigen or that have complementary activities.
[0167] Pharmaceutical compositions of the invention also can be
administered in combination therapy, i.e., combined with other
agents. For example, the combination therapy can include an
anti-IL-13 antibody of the present invention combined with at least
one other anti-inflammatory agent. Examples of therapeutic agents
that can be used in combination therapy are described in greater
detail below in the section on uses of the antibodies of the
invention.
[0168] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like that are physiologically compatible.
The carrier should be suitable for intravenous, intramuscular,
subcutaneous, parenteral, spinal or epidermal administration (e.g.,
by injection or infusion). Depending on the route of
administration, the active compound, i.e., antibody,
immunoconjuage, or bispecific molecule, may be coated in a material
to protect the compound from the action of acids and other natural
conditions that may inactivate the compound.
[0169] The pharmaceutical compounds of the invention may include
one or more pharmaceutically acceptable salts. A "pharmaceutically
acceptable salt" refers to a salt that retains the desired
biological activity of the parent compound and does not impart any
undesired toxicological effects (see e.g., Berge, S. M., et al.,
1977 J. Pharm. Sci. 66:1-19). Examples of such salts include acid
addition salts and base addition salts. Acid addition salts include
those derived from nontoxic inorganic acids, such as hydrochloric,
nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous
and the like, as well as from nontoxic organic acids such as
aliphatic mono- and di-carboxylic acids, phenyl-substituted
alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic
and aromatic sulfonic acids and the like. Base addition salts
include those derived from alkaline earth metals, such as sodium,
potassium, magnesium, calcium and the like, as well as from
nontoxic organic amines, such as N,N'-dibenzylethylenediamine,
N-methylglucamine, chloroprocaine, choline, diethanolamine,
ethylenediamine, procaine and the like.
[0170] A pharmaceutical composition of the invention also may
include a pharmaceutically acceptable anti-oxidant. Examples of
pharmaceutically acceptable antioxidants include: water soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium
bisulfate, sodium metabisulfite, sodium sulfite and the like;
oil-soluble antioxidants, such as ascorbyl palmitate, butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate, alpha-tocopherol, and the like; and metal chelating
agents, such as citric acid, ethylenediamine tetraacetic acid
(EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
[0171] Examples of suitable aqueous and nonaqueous carriers that
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0172] These compositions may also contain adjuvants such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of presence of microorganisms may be ensured
both by sterilization procedures, supra, and by the inclusion of
various antibacterial and antifungal agents, for example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be
desirable to include isotonic agents, such as sugars, sodium
chloride, and the like into the compositions. In addition,
prolonged absorption of the injectable pharmaceutical form may be
brought about by the inclusion of agents which delay absorption
such as, aluminum monostearate and gelatin.
[0173] Pharmaceutically acceptable carriers include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of sterile injectable solutions or dispersion. The use
of such media and agents for pharmaceutically active substances is
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into the
compositions.
[0174] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
liposome, or other ordered structure suitable to high drug
concentration. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), and suitable mixtures thereof. The proper fluidity can be
maintained, for example, by the use of a coating such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and by the use of surfactants. In many cases, one can
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought
about by including in the composition an agent that delays
absorption for example, monostearate salts and gelatin.
[0175] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by sterilization
microfiltration. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
methods of preparation are vacuum drying and freeze-drying
(lyophilization) that yield a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0176] The amount of active ingredient which can be combined with a
carrier material to produce a single dosage form will vary
depending upon the subject being treated, and the particular mode
of administration. The amount of active ingredient which can be
combined with a carrier material to produce a single dosage form
will generally be that amount of the composition which produces a
therapeutic effect. Generally, out of one hundred percent, this
amount will range from about 0.01 percent to about ninety-nine
percent of active ingredient, from about 0.1 percent to about 70
percent, or from about 1 percent to about 30 percent of active
ingredient in combination with a pharmaceutically acceptable
carrier.
[0177] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
subjects to be treated; each unit contains a predetermined quantity
of active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention are
dictated by and directly dependent on the unique characteristics of
the active compound and the particular therapeutic effect to be
achieved, and the limitations inherent in the art of compounding
such an active compound for the treatment of sensitivity in
individuals.
[0178] For administration of the antibody, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example dosages can be 0.3 mg/kg body weight,
1 mg/kg body weight, 3 mg/kg body weight, 5 mg/kg body weight or 10
mg/kg body weight or within the range of 1-10 mg/kg. An exemplary
treatment regime entails administration once per week, once every
two weeks, once every three weeks, once every four weeks, once a
month, once every 3 months or once every three to 6 months. Dosage
regimens for an anti-IL-13 antibody of the invention may include 1
mg/kg body weight or 3 mg/kg body weight by intravenous or
subcutaneous administration, with the antibody being given using
one of the following dosing schedules: e.g. every four weeks for
six dosages, then every three months; every three weeks; 3 mg/kg
body weight once followed by 1 mg/kg body weight every three
weeks.
[0179] In some methods, two or more monoclonal antibodies with
different binding specificities are administered simultaneously, in
which case the dosage of each antibody administered falls within
the ranges indicated. Antibody is usually administered on multiple
occasions. Intervals between single dosages can be, for example,
weekly, monthly, every three months or yearly. Intervals can also
be irregular as indicated by measuring blood levels of antibody to
the target antigen in the patient. In some methods, dosage is
adjusted to achieve a plasma antibody concentration of about 1-1000
.mu.g/ml and in some methods about 25-300 .mu.g/ml.
[0180] Alternatively, antibody can be administered as a sustained
release formulation, in which case less frequent administration is
required. Dosage and frequency vary depending on the half-life of
the antibody in the patient. In general, human antibodies show the
longest half-life, followed by humanized antibodies, chimeric
antibodies, and nonhuman antibodies. The dosage and frequency of
administration can vary depending on whether the treatment is
prophylactic or therapeutic. In prophylactic applications, a
relatively low dosage is administered at relatively infrequent
intervals over a long period of time. Some patients continue to
receive treatment for the rest of their lives. In therapeutic
applications, a relatively high dosage at relatively short
intervals is sometimes required until progression of the disease is
reduced or terminated or until the patient shows partial or
complete amelioration of symptoms of disease. Thereafter, the
patient can be administered a prophylactic regime.
[0181] Actual dosage levels of the active ingredients in the
pharmaceutical compositions of the present invention may be varied
so as to obtain an amount of the active ingredient which is
effective to achieve the desired therapeutic response for a
particular patient, composition, and mode of administration,
without being toxic to the patient. The selected dosage level will
depend upon a variety of pharmacokinetic factors including the
activity of the particular compositions of the present invention
employed, or the ester, salt or amide thereof, the route of
administration, the time of administration, the rate of excretion
of the particular compound being employed, the duration of the
treatment, other drugs, compounds and/or materials used in
combination with the particular compositions employed, the age,
sex, weight, condition, general health and prior medical history of
the patient being treated, and like factors well known in the
medical arts.
[0182] A "therapeutically effective dosage" of an anti-IL-13
antibody of the invention can results in a decrease in severity of
disease symptoms, an increase in frequency and duration of disease
symptom-free periods, or a prevention of impairment or disability
due to the disease affliction.
[0183] A composition of the present invention can be administered
by one or more routes of administration using one or more of a
variety of methods known in the art. As will be appreciated by the
skilled artisan, the route and/or mode of administration will vary
depending upon the desired results. Routes of administration for
antibodies of the invention include intravenous, intramuscular,
intradermal, intraperitoneal, subcutaneous, spinal or other
parenteral routes of administration, for example by injection or
infusion. The phrase "parenteral administration" as used herein
means modes of administration other than enteral and topical
administration, usually by injection, and includes, without
limitation, intravenous, intramuscular, intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal,
intraperitoneal, transtracheal, subcutaneous, subcuticular,
intraarticular, subcapsular, subarachnoid, intraspinal, epidural
and intrasternal injection and infusion.
[0184] Alternatively, an antibody of the invention can be
administered by a nonparenteral route, such as a topical, epidermal
or mucosal route of administration, for example, intranasally,
orally, vaginally, rectally, sublingually or topically.
[0185] The active compounds can be prepared with carriers that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0186] Therapeutic compositions can be administered with medical
devices known in the art. For example, in one embodiment, a
therapeutic composition of the invention can be administered with a
needleless hypodermic injection device, such as the devices shown
in U.S. Pat. No. 5,399,163; 5,383,851; 5,312,335; 5,064,413;
4,941,880; 4,790,824 or 4,596,556. Examples of well known implants
and modules useful in the present invention include: U.S. Pat. No.
4,487,603, which shows an implantable micro-infusion pump for
dispensing medication at a controlled rate; U.S. Pat. No.
4,486,194, which shows a therapeutic device for administering
medicants through the skin; U.S. Pat. No. 4,447,233, which shows a
medication infusion pump for delivering medication at a precise
infusion rate; U.S. Pat. No. 4,447,224, which shows a variable flow
implantable infusion apparatus for continuous drug delivery; U.S.
Pat. No. 4,439,196, which shows an osmotic drug delivery system
having multi-chamber compartments; and U.S. Pat. No. 4,475,196,
which shows an osmotic drug delivery system. These patents are
incorporated herein by reference. Many other such implants,
delivery systems, and modules are known to those skilled in the
art.
[0187] In certain embodiments, the human monoclonal antibodies of
the invention can be formulated to ensure proper distribution in
vivo. For example, the blood-brain barrier (BBB) excludes many
highly hydrophilic compounds. To ensure that the therapeutic
compounds of the invention cross the BBB (if desired), they can be
formulated, for example, in liposomes. For methods of manufacturing
liposomes, see, e.g., U.S. Pat. Nos. 4,522,811; 5,374,548; and
5,399,331. The liposomes may comprise one or more moieties which
are selectively transported into specific cells or organs, thus
enhance targeted drug delivery (see, e.g., V. V. Ranade, 1989 J.
Cline Pharmacol. 29:685). Exemplary targeting moieties include
folate or biotin (see, e.g., U.S. Pat. No. 5,416,016 to Low et
al.); mannosides (Umezawa et al., 1988 Biochem. Biophys. Res.
Commun. 153:1038); antibodies (P. G. Bloeman et al., 1995 FEBS
Lett. 357:140; M. Owais et al., 1995 Antimicrob. Agents Chemother.
39:180); surfactant protein A receptor (Briscoe et al., 1995 Am. J.
Physiol. 1233:134); p 120 (Schreier et al., 1994 J. Biol. Chem.
269:9090); see also K. Keinanen; M. L. Laukkanen, 1994 FEBS Lett.
346:123; J. J. Killion; I. J. Fidler, 1994 Immunomethods 4:273.
Uses and Methods of the Invention
[0188] The antibodies (and immunoconjugates and bispecific
molecules) of the present invention have in vitro and in vivo
diagnostic and therapeutic utilities. For example, these molecules
can be administered to cells in culture, e.g. in vitro or in vivo,
or in a subject, e.g., in vivo, to treat, prevent or diagnose a
variety of disorders. The term "subject" as used herein in intended
to include human and non-human animals. Non-human animals includes
all vertebrates, e.g., mammals and non-mammals, such as non-human
primates, sheep, dogs, cats, cows, horses, chickens, amphibians,
and reptiles. The methods are particularly suitable for treating
human patients having a disorder associated with aberrant IL-13
expression. When antibodies to IL-13 are administered together with
another agent, the two can be administered in either order or
simultaneously.
[0189] In one embodiment, the antibodies (and immunoconjugates and
bispecific molecules) of the invention can be used to detect levels
of IL-13, or levels of cells that contain IL-13. This can be
achieved, for example, by contacting a sample (such as an in vitro
sample) and a control sample with the anti-IL-13 antibody under
conditions that allow for the formation of a complex between the
antibody and IL-13. Any complexes formed between the antibody and
IL-13 are detected and compared in the sample and the control. For
example, standard detection methods, well known in the art, such as
ELISA and flow cytometic assays, can be performed using the
compositions of the invention.
[0190] Accordingly, in one aspect, the invention further provides
methods for detecting the presence of IL-13 (e.g., human IL-13
antigen) in a sample, or measuring the amount of IL-13, comprising
contacting the sample, and a control sample, with an antibody of
the invention, or an antigen binding portion thereof, which
specifically binds to IL-13, under conditions that allow for
formation of a complex between the antibody or portion thereof and
IL-13. The formation of a complex is then detected, wherein a
difference in complex formation between the sample compared to the
control sample is indicative of the presence of IL-13 in the
sample.
[0191] Also within the scope of the invention are kits consisting
of the compositions (e.g., antibodies, human antibodies,
immunoconjugates and bispecific molecules) of the invention and
instructions for use. The kit can further contain a least one
additional reagent, or one or more additional antibodies of the
invention (e.g., an antibody having a complementary activity which
binds to an epitope on the target antigen distinct from the first
antibody). Kits typically include a label indicating the intended
use of the contents of the kit. The term label includes any
writing, or recorded material supplied on or with the kit, or which
otherwise accompanies the kit.
[0192] The invention having been fully described, it is further
illustrated by the following examples and claims, which are
illustrative and are not meant to be further limiting. Those
skilled in the art will recognize or be able to ascertain using no
more than routine experimentation, numerous equivalents to the
specific procedures described herein. Such equivalents are within
the scope of the present invention and claims. The contents of all
references, including issued patents and published patent
applications, cited throughout this application are hereby
incorporated by reference.
EXAMPLES
Example 1
Generation of Human IL-13-Specific Antibodies from Immunized
Splenic Libraries
[0193] The RNA from spleen was used to generate a phage display
library of randomly assorted H and L chain variable domains in a
Fab phage display vector as described in U.S. Pat. No. 6,794,132.
The phage display library was subjected to five rounds of selection
using biotinylated hrIL-13 in a solution-phase equilibrium binding
protocol as described in the patent. The first four rounds of
selection employed hrIL-13 at 10.sup.-8 M and the last round of
selection employed hrIL-13 at 10.sup.-9 M. The final signal to
noise ratio determined by counting pfu's recovered in the presence
of antigen divided by pfu's recovered in the absence of antigen was
37 for this library, indicating that greater than 90% of the phage
selected were expressing antibodies that bound hrIL-13. The phage
display library was then subcloned into a plasmid vector for the
expression of soluble Fab as described in U.S. Pat. No. 6,794,132.
The subcloned is library comprises plasmid vectors in E. coli, each
plasmid encoding a monoclonal Fab fragment. The subclone library
was plated out and colonies representing individual clones were
picked to inoculate 96-well plates. After overnight growth, the
plate cultures were used to archive frozen cell banks for the
clones in 96-well plates and also to seed replica 96-well plates
which were induced to express monoclonal antibodies. The next day,
these 96-well plate cultures were subjected to detergent extraction
and purification to recover microgram quantities of antibodies. The
purified antibodies were treated to remove endotoxin and sterile
filtered terminally. ELISA assays were conducted using biotinylated
rhIL-13 coated on avidin plates to identify which wells contained
functional positives. Sandwich assays targeting the constant region
of the antibodies were used to determine antibody concentrations in
different wells. The 96-well plates containing the antibodies and
the assay data were assessed for biological activity. The clones of
interest in the 96-well frozen cell banks were then sequenced to
identify the ones expressing unique antibodies. Subsequently, the
frozen cell banks of these unique clones were used to seed small
scale shake flask cultures and grown overnight. Large scale flasks
were seeded using the overnight cultures and then induced to
express antibodies. The next day, the flask cultures were
mechanically homogenized and purified to yield milligram quantities
of antibodies. The purified Fabs were processed for endotoxin
removal and subjected to terminal sterile filtration. The
functional activity of these antibodies was demonstrated by ELISA
using biotinylated rhIL-13 coated on avidin plates. The antibody
concentrations were determined by absorbance measurement at 280 nm.
The purified Fabs were assessed for in vitro binding and activity
in a cell based assay.
Example 2
Quantitative Analysis of Binding Affinities: Determination of
Anti-Human IL-13 Fab Candidates
[0194] Surface plasmon resonance measurements quantifying the
interaction of anti-IL-13 Fabs with several hrIL-13 are performed
using the optical biosensor, BIAcore 2000. Specific binding of
IL-13 to a respective IL-13 Fab immobilized on a BIAcore chip can
be measured by following accumulation of the ligand on the
receptor. The microscopic association (k.sub.on) and dissociation
rates (k.sub.off) can be obtained directly from the mass
accumulation rates on the chip and are expressed in response units
(RUs). Anti-IL-13 Fab is immobilized on the chip surface via a
secondary anti-human L.kappa. antibody (Jackson Immunochemicals).
This capture antibody was covalently bound using the `Amine
coupling kit` (BIAcore, Cat. No. BR-1000-50) as recommended in the
manufacturers' protocols. 250 .mu.l of varying concentrations of
hrIL-13 was injected at flow rate of 20 .mu.l/min and the kinetic
trace was recorded. The chip surface was regenerated by two acid
wash steps using 100 mM HCl and injecting 10 .mu.l with a flow rate
of 20 .mu.l/min. This treatment leads to dissociation of the Fab
IL-13 complex due to reversible acid denaturation. No significant
loss of binding activity was observed when the antibody was
reinjected for a subsequent run. The kinetic traces were evaluated
with the BIAcore software applying the 1:1 Langmuir association
model
The summarized affinity data on human IL-13 is shown in Table 1
herein.
TABLE-US-00001 TABLE 1 Fab KD [pM] human IL-13 01471/G6 100 .+-. 2
03161/H2 197 .+-. 12 01951/G12 480 .+-. 68 01771/E10 343 .+-.
54
Example 3
Conversion into the IgG Format
[0195] Antibody DNA sequencing templates were purified from 3 ml
cultures using QIAprep minipreps (Qiagen Inc.). Templates were
sequenced using an Applied Biosystems 3100 Avant Genetic Analyzer
according to manufacturer's specifications. The heavy and kappa
chain variable regions of selected clones were separately amplified
from the sequencing templates by PCR, purified by agarose gel
electrophoresis, and excised and purified from the gel. Plasmids
encoding the V.sub.H and V.sub.L were cloned into expression
cassettes for human kappa light and human IgG.sub.1 heavy chains.
The Sp2/0 parental cell line was transfected with two vectors, one
for the light and one for heavy chain vectors. Transfected cells
were selected and amplified using G418 and methotrexate
respectively resulting in the emergence of resistant, amplified
cell pools producing the antibodies with titers ranging from 5 mg/L
to 30 mg/L. Dilution cloning was then employed, resulting in the
isolation of 127 viable clones from six 96-well plates. Emerging
cell lines were then tested for productivity in a fed-batch shaker
format. Stability testing to confirm the stable integration of the
expression constructs and robust expression of product were also
performed over a 90-day period. Northern blots exhibited single,
full length RNAs with equal band intensities, indicating similar
expression levels for both the heavy chain and light chain.
Example 4
In Vitro Characterization of Anti-IL-13 Full Antibodies in a Cell
Based Assay
[0196] IL-13 is a potent inducer of eotaxin release from human lung
fibroblasts. The ability of the antibodies to neutralize the
bioactivity of IL-13 was assessed in an IL-13-induced eotaxin
release assay using human lung fibroblasts. Briefly, cells
(2.times.10.sup.4 cells per well in a volume of 100 .mu.l) were
plated out in each well of a 96 well tissue culture plate. The
cells were stimulated with a concentration of IL-13 conferring 80%
of the maximal eotaxin release, which was pre-determined for each
batch of cells using a standard curve of 0-100 ng/ml IL-13. Varying
concentrations of the antibodies were co-applied to the cells. The
cells were allowed to incubate for 24 h at 37.degree. C., 5%
CO.sub.2 and the culture media was harvested and stored at
-20.degree. C. until required. Eotaxin levels within the media were
measured by specific ELISA (R&D systems) where the sensitivity
of assay was between 15-1000 pg/ml.
[0197] The anti-IL-13 Fabs were thereby analyzed with respect to
EC.sub.50 as described above and shown in Table 2.
TABLE-US-00002 TABLE 2 Antibody EC.sub.50 [nM] human IL-13 01471/G6
1.23 .+-. 0.4 03161/H2 0.95 .+-. 0.2 01951/G12 0.33 .+-. 0.1
01771/E10 1.71 .+-. 0.5
Example 5
Sequence Analysis of the Anti-IL-13 Antibodies
[0198] The nucleotide sequences of the heavy and light chain
variable regions (V.sub.H and V.sub.L) of all antibodies were
determined. Amino acid sequences of the complementarity determining
regions (CDRs) are listed in Table 3 and 4 herein. The CDRs
according Kabat definition (E. Kabat et al, 1991, Sequences of
Proteins of Immunological Interest, 5th edition, Public Health
Service, National Institute of Health, Bethesda, Md., are listed in
Table 3a and 4a.
TABLE-US-00003 TABLE 3 SEQ ID No. SEQ ID No. SEQ ID No. Antibody
HCDR1 HCDR1 HCDR2 HCDR2 HCDR3 HCDR3 01471/G6 GFTFSNYG 1 IWYDGSN 3
VKGSGDIP 4 03161/H2 GFTFSNYG 1 IWYDGSN 3 VKGSGDIP 4 01951/G12
GFTFSSYG 2 IWYDGSN 3 ARLWFGDLD 5 01771/E10 GFTFSSYG 2 IWYDGSN 3
ARLWFGDLD 5
TABLE-US-00004 TABLE 3a SEQ ID No. SEQ ID No. SEQ ID No. Antibody
HCDR1 HCDR1 HCDR2 HCDR2 HCDR3 HCDR3 01471/G6 NYGMH 6
IIWYDGSNKYYADSVKG 8 GSGDIPFDY 9 03161/H2 NYGMH 6 IIWYDGSNKYYADSVKG
8 GSGDIPFDY 9 01951/G12 SYGMH 7 IIWYDGSNKYYADSVKG 8 LWFGDLDAFDI 10
01771/E10 SYGMH 7 IIWYDGSNKYYADSVKG 8 LWFGDLDAFDI 10
TABLE-US-00005 TABLE 4 SEQ ID No. SEQ ID No. SEQ ID No. Antibody
LCDR1 LCDR1 LCDR2 LCDR2 LCDR3 LCDR3 01471/G6 QSVSSY 11 DA 12
HQRSHWPPI 13 03161/H2 QSVSSY 11 DA 12 HQRSHWPPI 13 01951/G12 QSVSSY
11 DA 12 QQRSSWPPV 14 01771/E10 QSVSSY 11 DA 12 HQRSSWPPI 15
TABLE-US-00006 TABLE 4a SEQ ID No. SEQ ID No. SEQ ID No. Antibody
LCDR1 LCDR1 LCDR2 LCDR2 LCDR3 LCDR3 01471/G6 RASQSVSSYLA 16
DASNRAAT 19 HQRSHWPPIFT 20 03161/H2 RASQSVSSYLA 16 DASNRAT 19
HQRSHWPPIFT 20 01951/G12 RAGQSVSSYLV 17 DASNRAT 19 QQRSSWPPVYT 21
01771/E10 RASQSVSSYLA 18 DASNRAT 19 HQRSSWPPIFT 22
The sequences of the antibodies of the previous tables, including
framework regions, are shown below. The full IgG1 antibody light
and heavy chain constant regions are also shown below,
incorporating, as an example, the variable regions of antibody
01951/G12 (emboldened).
01471/G6 Antibody Sequence
(i) HC Variable Region
[0199] The HC variable amino acid sequence for 01471/G6 is shown in
SEQ ID NO: 23 and is encoded by the nucleotide sequence shown in
SEQ ID NO: 24
TABLE-US-00007 E V Q L V E S G G G V V Q P G R S L R L (SEQ ID NO:
23) gaagtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactc 60
(SEQ ID NO: 24) S C A A S G F T F S N Y G M H W V R Q A
tcctgtgcagcgtctggattcaccttcagtaactatggcatgcactgggtccgccaggct 120 P
G K G L E W V A I I W Y D G S N K Y Y
ccaggcaaggggctggagtgggtggcaattatatggtatgatggaagtaataaatactat 180 A
D S V K G R F T I S R D N S K N T L Y
gcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtat 240 L
Q M N S L R A E D T A V Y Y C V K G S
ctgcaaatgaacagtctgagagccgaggacacggctgtgtattactgtgtgaaaggatct 300 G
D I P F D Y W G Q G T L V T
ggggatattccctttgactactggggccagggaaccctggtcacc 345
(ii) LC Variable Region
[0200] The LC variable amino acid sequence for 01471/G6 is shown in
SEQ ID NO: 25 and is encoded by the nucleotide sequence shown in
SEQ ID NO: 26
TABLE-US-00008 E I V L T Q S P A T L S S S P G E R A T (SEQ ID NO:
25) gaaattgtgttgacgcagtctccagccaccctgtcttcgtctccaggggaaagagccacc 60
(SEQ ID NO: 26) L S C R A S Q S V S S Y L A W Y Q Q K P
ctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacct 120 G
Q A P R L L I Y D A S N R A T G I P A
ggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagcc 180 R
F S G S G S G T D F T L T I S S L E P
aggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcct 240 E
D F A V Y Y C H Q R S H W P P I F T F
gaagattttgcagtctattactgtcatcagcgtagccactggcctcccatattcactttc 300 G
P G T ggccctgggacc 312
03161/H2 Antibody
(i) HC Variable Region
[0201] The HC variable amino acid sequence for 03161/H2 is shown in
SEQ ID NO: 27 and is encoded by the nucleotide sequence shown in
SEQ ID NO: SEQ ID No. 28
TABLE-US-00009 E V Q L V E S G G G V V Q P G R S L R L (SEQ ID NO:
27) gaagtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactc 60
(SEQ ID NO: 28) S C A A S G F T F S N Y G M H W V R Q A
tcctgtgcagcgtctggattcaccttcagtaactatggcatgcactgggtccgccaggct 120 P
G K G L E W V A I I W Y D G S N K Y Y
ccaggcaaggggctggagtgggtggcaattatatggtatgatggaagtaataaatactat 180 A
D S V K G R F T I S R D N S K N T L Y
gcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtat 240 L
Q M N S L R A E D T A V Y Y C V K G S
ctgcaaatgaacagtctgagagccgaggacacggctgtgtattactgtgtgaaaggatct 300 G
D I P F D Y W G Q G T L V T
ggggatattccctttgactactggggccagggaaccctggtcacc 345>
(ii) LC Variable Region
[0202] The LC variable amino acid sequence for 03161/H2 is shown in
SEQ ID NO: 29 and is encoded by the nucleotide sequence shown in
SEQ ID NO: 30
TABLE-US-00010 E I V L T Q S P A T L S S S P G E R A T (SEQ ID NO:
29) gaaattgtgttgacgcagtccccagccaccctgtcttcgtctccaggggaaagagccacc 60
(SEQ ID NO: 30) L S C R A S Q S V S S Y L A W Y Q Q K P
ctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacct 120 G
Q A P R L L I Y D A S N R A T G T P A
ggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcaccccagcc 180 R
F S G S G S G T D F T L T I S S L E P
aggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcct 240 E
D F A V Y Y C H Q R S H W P P I F T F
gaagattttgcagtctattactgtcatcagcgtagccactggcctcccatattcactttc 300 G
P G T ggccctgggacc 312
01951/G12 Antibody Sequence
(i) HC Variable Region
[0203] The HC variable amino acid sequence for 01951/G12 is shown
in SEQ ID NO: 31 and is encoded by the nucleotide sequence shown in
SEQ ID NO: 32
TABLE-US-00011 E V Q L V E S G G G V V Q P G R S L R L (SEQ ID NO:
31) gaagtgcagctggtggagtctgggggaggcgtggtccagcctgggaggtccctgagactc 60
(SEQ ID NO: 32) S C A A S G F T F S S Y G M H W V R Q A
tcctgtgcagcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggct 120 P
G K G L E W V A I I W Y D G S N K Y Y
ccaggcaaggggctggagtgggtggcaattatatggtatgatggaagtaataaatactat 180 A
D S V K G R F T I S R D N S K N T L Y
gcggactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtat 240 L
Q M N S L R A E D T A V Y Y C A R L W
ctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaggctatgg 300 F
G D L D A F D I W G Q G T M V T
ttcggggacttagatgcttttgatatctggggccaagggacaatggtcacc 351
(ii) LC Variable Region
[0204] The LC variable amino acid sequence for 01951/G12 is shown
in SEQ ID NO: 33 and is encoded by the nucleotide sequence shown in
SEQ ID NO: 34
TABLE-US-00012 E I V L T Q S P A T L S L S F G E R A I (SEQ ID NO:
33) gaaattgtgttgacgcagtctccagccaccctgtctttgtctccaggggaaagagccatc 60
(SEQ ID NO: 34) L S C R A G Q S V S S Y L V W Y Q Q K P
ctctcctgcagggccggtcagagtgttagcagttacttagtctggtaccaacagaaacct 120 G
Q A P R L L I Y D A S N R A T G I P A
ggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagcc 180 R
F S G S G S G T D F T L T I S S L E P
aggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcct 240 E
D F A V Y Y C Q Q R S S W P P V Y T F
gaagattttgcagtttattactgtcagcagcgcagcagctggcctccggtgtacactttt 300 G
Q G T ggccaggggacc 312
01771/E10 Antibody Sequence
(i) HC Variable Region
[0205] The HC variable amino acid sequence for 01771/E10 is shown
in SEQ ID NO: 35 and is encoded by the nucleotide sequence shown in
SEQ ID NO: 36
TABLE-US-00013 Q V Q L V Q S G G G V V Q P G R S L R L (SEQ ID NO:
35) caggtgcagctggtgcagtctgggggaggcgtggtccagcctgggaggtccctgagactc 60
(SEQ ID NO: 36) S C A A S G F T F S S Y G M H W V R Q A
tcctgtgcggcgtctggattcaccttcagtagctatggcatgcactgggtccgccaggct 120 P
G K G L E W V A I I W Y D G S N K Y Y
ccaggcaaggggctggagtgggtggcaattatatggtatgatggaagtaataaatactat 180 A
D S V K G R F T I S R D N S K N T L Y
gcggactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctatat 240 L
Q M N S L R A E D T A V Y Y C A R L W
ctacaaatgaacagcctgagagccgaggacacggctgtgtattactgtgcgaggctatgg 300 F
G D L D A F D I W G Q G T M V T
ttcggggacttagatgcttttgatatctggggccaagggacaatggtcacc 351
(ii) LC Variable Region
[0206] The LC variable amino acid sequence for 01771/E10 is shown
in SEQ ID NO: 37 and is encoded by the nucleotide sequence shown in
SEQ ID NO: 38
TABLE-US-00014 E I V L T Q S P A T L S L S P G E R A T (SEQ ID NO:
37) gaaattgtgttgacgcagtctccagccaccctgtctttgtctccaggggaaagagccacc 60
(SEQ ID NO: 38) L S C R A S Q S V S S Y L A W Y Q Q K P
ctctcctgcagggccagtcagagtgttagcagctacttagcctggtaccaacagaaacct 120 G
Q A P R L L I Y D A S N R A T G I P A
ggccaggctcccaggctcctcatctatgatgcatccaacagggccactggcatcccagcc 180 R
F S G S G S G T D F T L T I S S L E P
aggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctagagcct 240 E
D F A V Y Y C H Q R S S W P P I F T F
gaagattttgcggtttattactgtcatcagcgtagcagctggcccccgatattcactttc 300 G
P G T ggccctgggacc 312
Full Antibody IgG1 Light Chain Sequence Incorporating the Variable
Region Of Antibody 01951/G12 (Emboldened)
[0207] The LC amino acid sequence is shown in SEQ ID NO: 39 and is
encoded by the nucleotide sequence of SEQ ID NO: 40
TABLE-US-00015 M S V L T Q V L A L L L L W L T G (SEQ ID NO: 39) 1
ATGAGTGTGC TCACTCAGGT CCTGGCGTTG CTGCTGCTGT GGCTTACAGG (SEQ ID NO:
40) T R C E I V L T Q S P A T L S L S 51 TACGCGTTGT GAAATTGTGT
TGACGCAGTC TCCAGCCACC CTGTCTTTGT P G E R A I L S C R A G Q S V S
101 CTCCAGGGGA AAGAGCCATC CTCTCCTGCA GGGCCGGTCA GAGTGTTAGC S Y L V
W Y Q Q K P G Q A P R L L 151 AGTTACTTAG TCTGGTACCA ACAGAAACCT
GGCCAGGCTC CCAGGCTCCT I Y D A S N R A T G I P A R F S G 201
CATCTATGAT GCATCCAACA GGGCCACTGG CATCCCAGCC AGGTTCAGTG S G S G T D
F T L T I S S L E P 251 GCAGTGGGTC TGGGACAGAC TTCACTCTCA CCATCAGCAG
CCTAGAGCCT E D F A V Y Y C Q Q R S S W P P V 301 GAAGATTTTG
CAGTTTATTA CTGTCACCAG CGCAGCAGCT GGCCTCCGGT Y T F G Q G T K L E I K
R T V A A 351 GTACACTTTT GGCCAGGGGA CCAAGCTTGA AATCAAACGA
ACTGTGGCTG P S V F I F P P S D E Q L K S G 401 CACCATCTGT
CTTCATCTTC CCGCCATCTG ATGAGCAGTT GAAATCTGGA T A S V V C L L N N F Y
P R E A K 451 ACTGCCTCTG TTGTGTGCCT GCTGAATAAC TTCTATCCCA
GAGAGGCCAA V Q W K V D N A L Q S G N S Q E S 501 AGTACAGTGG
AAGGTGGATA ACGCCCTCCA ATCGGGTAAC TCCCAGGAGA V T E Q D S K D S T Y S
L S S T 551 GTGTCACAGA GCAGGACAGC AAGGACAGCA CCTACAGCCT CAGCAGCACC
L T L S K A D Y E K H K V Y A C E 601 CTGACGCTGA GCAAAGCAGA
CTACGAGAAA CACAAAGTCT ACGCCTGCGA V T H Q G L S S P V T K S F N R G
651 AGTCACCCAT CAGGGCCTGA GCTCGCCCGT CACAAAGAGC TTCAACAGGG E C *
701 GAGAGTGTTA G
Full Antibody IgG1 Heavy Chain Sequence Incorporating the Variable
Region of Antibody 01951/G12 (Emboldened)
[0208] The HC amino acid sequence is shown in SEQ ID NO: 41 and is
encoded by the nucleotide sequence of SEQ ID NO: 42
TABLE-US-00016 M A W V W T L P F L M A A A Q S V (SEQ ID NO:41) 1
ATGGCTTGGG TGTGGACCTT GCCATTCCTG ATGGCAGCTG CCCAAAGTGT (SEQ ID
NO:42) Q A E V Q L V E S G G G V V Q P G 51 CCAGGCAGAA GTGCAGCTGG
TGGAGTCTGG GGGAGGCGTG GTCCAGCCTG R S L R L S C A A S G F T F S S
101 GGAGGTCCCT GAGACTCTCC TGTGCAGCGT CTGGATTCAC CTTCAGTAGC Y G M H
W V R Q A P G K G L E W V 151 TATGGCATGC ACTGGGTCCG CCAGGCTCCA
GGCAAGGGGC TGGAGTGGGT A I I W Y D G S N K Y Y A D S V K 201
GGCAATTATA TGGTATGATG GAAGTAATAA ATACTATGCG GACTCCGTGA G R F T I S
R D N S K N T L Y L 251 AGGGCCGATT CACCATCTCC AGAGACAATT CCAAGAACAC
GCTGTATCTG Q M N S L R A E D T A V Y Y C A R 301 CAAATGAACA
GCCTGAGAGC CGAGGACACG GCTGTGTATT ACTGTGCGAG L W F G D L D A F D I W
G Q G T M 351 GCTATGGTTC GGGGkCTTAG ATGCTTTTGA TATCTGGGGC
CAAGGGACAA V T V S S A S T K G P S V F P L 401 TGGTCACCGT
CTCCTCAGCC TCCACCAAGG GCCCATCGGT CTTCCCCCTG A P S S K S T S G G T A
A L G C L 451 GCACCCTCCT CCAAGAGCAC CTCTGGGGGC ACAGCGGCCC
TGGGCTGCCT V K D Y F P E P V T V S W N S G A 501 GGTCAAGGAC
TACTTCCCCG AACCGGTGAC GGTGTCGTGG AACTCAGGCG L T S G V H T F P A V L
Q S S G 551 CCCTGACCAG CGGCGTGCAC ACCTTCCCGG CTGTCCTACA GTCCTCAGGA
L Y S L S S V V T V P S S S L G T 601 CTCTACTCCC TCAGCAGCGT
CGTGACCGTG CCCTCCAGCA GCTTGGGCAC Q T Y I C N V N H K P S N T K V D
651 CCAGACCTAC ATCTGCAACG TGAATCACAA GCCCAGCAAC ACCAAGGTGG K R V E
P K S C D K T H T C P P 701 ACAAGAGAGT TGAGCCCAAA TCTTGTGACA
AAACTCACAC ATGCCCACCG C P A P E L L G G P S V F L F P P 751
TGCCCAGCAC CTGAACTCCT GGGGGGACCG TCAGTCTTCC TCTTCCCCCC K P K D T L
M I S R T P E V T C V 801 AAAACCCAAG GACACCCTCA TGATCTCCCG
GACCCCTGAG GTCACATGCG V V D V S H E D P E V K F N W Y 851
TGGTGGTGGA CGTGAGCCAC GAAGACCCTG AGGTCAAGTT CAACTGGTAC V D G V E V
H N A K T K P R E E Q 901 GTGGACGGCG TGGAGGTGCA TAATGCCAAG
ACAAAGCCGC GGGAGGAGCA Y N S T Y R V V S V L T V L H Q D 951
GTACAACAGC ACGTACCGTG TGGTCAGCGT CCTCACCGTC CTGCACCAGG W L N G K E
Y K C K V S N K A L 1001 ACTGGCTGAA TGGCAAGGAG TACAAGTGCA
AGGTCTCCAA CAAAGCCCTC P A P I E K T I S K A K G Q P R E 1051
CCAGCCCCCA TCGAGAAAAC CATCTCCAAA GCCAAAGGGC AGCCCCGAGA P Q V Y T L
P P S R E E M T K N Q 1101 ACCACAGGTG TACACCCTGC CCCCATCCCG
GGAGGAGATG ACCAAGAACC V S L T C L V K G F Y P S D I A 1151
AGGTCAGCCT GACCTGCCTG GTCAAAGGCT TCTATCCCAG CGACATCGCC V E W E S N
G Q P E N N Y K T T P 1201 GTGGAGTGGG AGAGCAATGG GCAGCCGGAG
AACAACTACA AGACCACGCC P V L D S D G S F F L Y S K L T V 1251
TCCCGTGCTG GACTCCGACG GCTCCTTCTT CCTCTATAGC AAGCTCACCG D K S R W Q
Q G N V F S C S V M 1301 TGGACAAGAG CAGGTGGCAG CAGGGGAACG
TCTTCTCATG CTCCGTGATG H E A L H N H Y T Q K S L S L S P 1351
CATGAGGCTC TGCACAACCA CTACACGCAG AAGAGCCTCT CCCTGTCCCC G K * 1401
GGGTAAATGA
* * * * *
References